US20230136088A1

US20230136088A1 - miRNA-193a for Promoting Immunogenic Cell Death

Info

Publication number: US20230136088A1
Application number: US17/904,864
Authority: US
Inventors: Sanaz YAHYANEJAD; Bryony TELFORD; Marion VAN DEN BOSCH; Mir Farshid ALEMDEHY; Matheus Maria De Gunst; Laurens Adrianus Hendricus VAN PINXTEREN; Roeland Quirinus Jozef Schaapveld; Michel JANICOT
Original assignee: Interna Technologies BV
Current assignee: Interna Technologies BV
Priority date: 2020-02-28
Filing date: 2021-02-26
Publication date: 2023-05-04
Also published as: AU2021227002A1; CA3167367A1; JP2023515010A; WO2021170786A1; CN115461461A; EP4110917A1

Abstract

The invention relates to the use of miRNA-193a for regulating gene expression, particularly it relates to the use of miRNA-193a as a CRT agonist, promoting the cell surface expression of CRT. This allows the advantageous treatment of cancers without or with low surface expression of CRT. The invention further relates to compositions comprising the miRNA for use in such treatment.

Description

FIELD OF THE INVENTION

The invention relates to the use of miRNA-193a for regulating gene expression, particularly it relates to the use of miRNA-193a as a means to achieve immunogenic cell death in cancer cells. This allows the advantageous treatment of various cancer types, particularly of cancers low in expression of calreticulin. The invention further relates to compositions comprising the miRNA for use in treating such conditions.

BACKGROUND ART

MicroRNAs (miRNAs) are naturally occurring single-stranded, non-coding small RNA molecules that control gene expression by binding to complementary sequences in their target mRNAs, thereby inhibiting translation or inducing mRNA degradation. miRNAs have recently emerged as key regulators of gene expression during development and are frequently misexpressed in human disease states, for example in cancer. In fact, miRNAs can be used to silence specific cancer genes. Several miRNAs are reported to be effective modulators of cancer. For example, miRNA-193a has been described as effective in treating melanoma (WO2012005572).
Immunogenic cell death (ICD) is a unique class of regulated cell death that activates the adaptive immune system against dead-cell-associated antigens. ICD is hence capable of eliciting antigen-specific immune responses (see Galluzzi, Lorenzo, and Ilio Vitale. 2018. “Molecular Mechanisms of Cell Death: Recommendations of the Nomenclature Committee on Cell Death 2018,” 486-541; and Kroemer, Guido, Lorenzo Galluzzi, Oliver Kepp, and Laurence Zitvogel. 2013. “Immunogenic Cell Death in Cancer Therapy.”) Considering the potential of ICD in prompting an adaptive immune response against dying cells, induction of ICD in tumor cells eventually culminates in an enhanced anti-tumor immune-cell-mediated cytotoxicity, and more importantly formation of an immunological memory against the tumor antigens. Therefore, ICD induction in tumor cells is considered a potent immunotherapeutic approach to activate antitumor immunity (see Obeid, Michel, Antoine Tesniere, et al. 2007. “Calreticulin Exposure Dictates the Immunogenicity of Cancer Cell Death” 13 (1): 54-61; and Palucka, Karolina, and Jacques Banchereau. 2012. “NIH Public Access.” Nature Reviews Cancer 12 (4): 265-77).
ICD is associated with a series of immunogenic molecular patterns known as damage associated molecular patterns (DAMPs). DAMPs are molecules that are secreted, released, or surface exposed as a result of premortem endoplasmic reticulum stress and autophagy. These molecules can then function as either adjuvant or danger signals to activate the immune system. DAMPs include translocation of heat shock proteins (HSPs) (including HSP70 and HSP90) and chaperone calreticulin (CRT) to the outer surface of plasma membrane, and release of soluble mediators, including high-mobility group box 1 protein (HMGB1) and adenosine triphosphate (ATP) (see Krysko, Dmitri V, Abhishek D Garg, et al., 2012. Nature Reviews Cancer 12 (12): 860-75; Casares, Noelia, Marie O Pequignot e al., 2005. “Caspase-Dependent Immunogenicity of Doxorubicin-Induced Tumor Cell Death” 202 (12)). CRT, ATP, and HMGB1 bind to dendritic cell (DC) receptors CD91, P2RX7, and TLR4, respectively. These bindings facilitate the maturation of DCs, the recruitment of mature DCs into the tumor microenvironment (stimulated by ATP), the engulfment of tumor antigens by DCs (stimulated by CRT), and optimal antigen presentation of tumor-cell-associated antigens to T cells (stimulated by HMGB1), leading to T cell proliferation and activation. Altogether, these processes result in a potent anti-tumor immune response involving cytotoxic T cells (see Kroemer et al., cited above)
Selected approved chemotherapeutic agents including, but not limited to, the DNA alkylating agent cyclophosphamide, the anthracyclines doxorubicin and mitoxantrone, and the platinum derivative oxaliplatin have the ability to trigger ICD, hence activating anticancer immune responses. These drugs are used to treat different types of hematological and solid malignancies, including breast and ovarian, colorectal, and prostate cancers, and myeloid and lymphoid leukemias (see Vacchelli, Erika, Laura Senovilla, et al. 2013. “Trial Watch Chemotherapy with Immunogenic Cell Death Inducers Trial Watch.”; and Garg, Abhishek D, Sanket More et al., “Trial Watch: Immunogenic Cell Death Induction by Anticancer Chemotherapeutics” 6 (12): 1-18.) Moreover, there are several ongoing clinical trials assessing the off-label potential of conventional chemotherapy in eliciting immunogenicity (see Showalter, Anne, Arati Limaye, et al., Cytokine 97 (May): 123-32.) However, only a limited number of cell death inducers can elicit bona fide ICD, and this capacity cannot be predicted on the basis of structural or functional similarities (see Garg et al., cited above).
An approach to evaluate the ability of a specific stimulus to cause ICD relies on vaccination experiments. In this setting, immunocompetent mice are vaccinated with dead syngeneic tumor cells, killed in vitro by a cytotoxic agent of choice. Later, vaccinated mice are challenged with living tumor cells of the same type. The proportion of mice that do not develop tumors reflects the degree of immunogenicity of cell death as induced by the cytotoxic agent under evaluation. Although they constitute a common approach for the detection of ICD, vaccination assays relying on immunocompetent mice and syngeneic cancer cells are intrinsically incompatible with large screening. To circumvent this issue, and to enable quick capacity evaluation of different cytotoxic agents for ICD induction, various in vitro techniques that allow for detection of one or more ICD manifestations have been developed. Among them are methods to detect surrogate ICD biomarkers including cell surface CRT exposure, ATP secretion and HMGB1 release (see Kepp, Oliver, Laura Senovilla, Ilio Vitale, et al., 2014. “Consensus Guidelines for the Detection of Immunogenic Cell Death.”)
Calreticulin (CRT) is a protein that usually resides in the lumen of the endoplasmatic recitulum, but which can be exposed on the surface of cancer cells (Zitvogel et al., Clin. Cancer Res., 2010, DOI: 10.1158/1078-0432.CCR-09-2891). It is not a universal biomarker, and not all cancers express calreticulin on their surfaces. For instance, Harada et al. (Oncol. Lett. 2017, doi: 10.3892/01.2017.6062) found that in tissue samples taken from 111 oral squamous cell carcinoma (OSCC) patients high expression of CRT was observed in 44 patients (39.6%), whereas low expression was observed in 67 patients (60.4%), as determined by immunohistochemistry. Cancers with low CRT were reported to be associated with increased overall survival probability (Harada, see above).
Cancers with low CRT expression are refractory to the immune-mediated component of existing anticancer therapies. When elicited by chemotherapeutics such as anthracyclines, the CRT exposure pathway is activated by pre-apoptotic endoplasmatic reticulum (ER) stress and the phosphorylation of the eukaryotic translation initiation factor eIF2α by the kinase PERK, followed by caspase-8-mediated proteolysis of the ER-sessile protein BAP31, activation of the pro-apoptotic proteins Bax and Bak, anterograde transport of CRT from the ER to the Golgi apparatus and exocytosis of CRT-containing vesicles, finally resulting in CRT translocation onto the plasma membrane surface. Interruption of this complex pathway abolishes CRT exposure, which annihilates the immunogenicity of apoptosis.
Phosphatase and tensin homolog (PTEN) is 47-kDa protein and was first identified as a candidate tumour suppressor gene in 1997 after its positional cloning from a region of chromosome 10q23 known to exhibit loss in a wide spectrum of tumour types. Since then, mutations of PTEN have been detected in a variety of human cancers including breast, thyroid, glioblastoma, endometrial, and prostate cancer, and melanoma. Inherited mutations in this gene also predispose carriers to develop Cowden's disease, a heritable cancer risk syndrome, and several related conditions. PTEN is classified as a tumour suppressor because in various cancers its activity is lost by deletion, mutation, or through epigenetic changes. Molecular mechanistic studies of PTEN have provided insight into the basis for its involvement in tumour suppression. The PTEN protein has both protein phosphatase and lipid phosphatase activity. Although the tumour suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell-cycle regulation and inhibition of cell invasion in vitro has been suggested as well. Loss of PTEN function seems to be responsible for many of the phenotypic features of PTEN-deficient melanoma, thus PTEN may serve as a potential target for drug development. Even when mutation of PTEN has minimal effects, it frequently contributes to tumorigenesis in the context of other genetic alterations (Aguissa-Toure et al., Cellular and Molecular Life Sciences 69: 1475-1491 (2012)).
PTEN agonists are known in the art, and their use in treating cancer has been described (WO2009126842). Their activity can stem from inhibition of mTOR. Known PTEN agonists include rapamycin (sirolimus) and its chemical analogues such as CCI-779 (temsirolimus), and RAD-001 (everolimus). Many PTEN agonists are small molecules (i.e., a compound having relatively low molecular weight, most often less than 500 or 600 kDa, or about 1000 kDa in the case of a macrolide such as rapamycin). Other agonists include monoclonal antibodies, and zinc finger proteins or nucleic acids encoding the same, engineered to bind to and activate transcription of PTEN (see WO 00/00388). Other PTEN agonists are described in US20070280918. Exemplary sequences for human PTEN and mTOR(FRAPI) are assigned UniProtKB/Swiss-Prot accession numbers P60484 and P42345.
A disadvantage of PTEN agonists is that they are associated with several adverse effects. For example, the PTEN agonist sirolimus is commonly (over 30% occurrence) associated with effects as diverse as peripheral edema, hypercholesterolemia, abdominal pain, headache, nausea, diarrhea, pain, constipation, hypertriglyceridemia, hypertension, increased creatinine, fever, urinary tract infection, anemia, arthralgia, and thrombocytopenia, in addition to diabetes-like symptoms, and even an increased risk for contracting skin cancers from exposure to UV radiation (see “Rapamune Prescribing Information”, United States Food and Drug Administration, Wyeth Pharmaceuticals, Inc. May 2015). The PTEN agonist temsirolimus is associated with fatigue, skin rash, mucositis, decreased haemoglobin, and decreased lymphocytes (Bellmunt et al., Annals of Oncology, 2008 DOI: 10.1093/annonc/mdn066).
Accordingly, there is an ongoing need for alternative and improved CRT agonists, or methods for increasing expression or presentation of CRT on the cell surface of cancer cells. There is an ongoing need for alternative and improved PTEN agonists. There is an ongoing need for improved microRNA therapies for tumours, as well as an ongoing need for deeper mechanistic insight into microRNA treatment of tumours, which can open up new strategies for treatment. There is an ongoing need for means to restore impaired immunogenic cell death pathways, and to promote immunogenic cell death of cancer cells.

SUMMARY OF THE INVENTION

The invention provides a miRNA-193a or a source thereof, for use in treating a condition associated with low expression of calreticulin (CRT). Preferably the miRNA-193a is a CRT agonist. Preferably the miRNA-193a is a miRNA-193a molecule, an isomiR, or a mimic thereof, wherein it is preferably an oligonucleotide with a seed sequence comprising at least 6 of the 7 nucleotides of the seed sequence represented by SEQ ID NO: 22. Preferably the source of a miRNA is a precursor of a miRNA and is a nucleic acid of at least 50 nucleotides in length. Preferably, said miRNA shares at least 70% sequence identity with any one of SEQ ID NOs: 56, 121, or 122, and/or said miRNA is from 15-30 nucleotides in length, and/or said source of a miRNA is a precursor of said miRNA and shares at least 70% sequence identity with any one of SEQ ID NOs: 5 or 13. Preferably, the condition associated with low CRT expression is a low-CRT cancer. Preferably, the low-CRT cancer is a low-CRT sarcoma, brain cancer, head and neck cancer, breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, ovarian cancer, melanoma, pancreatic cancer, thyroid cancer, hamartoma, tumour of the haematopoietic and lymphoid malignancy, or prostate cancer. Preferably, the miRNA-193a modulates expression of a gene selected from the group consisting of CRT, HMGB1, RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1, preferably from the group consisting of RPS6KB2, KRAS, PDGFRB, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, MCL1, more preferably selected from PDPK1 or INPPL1.
The invention further provides a composition comprising a miRNA-193a or a source thereof as defined above, for use as defined above. Preferably the composition further comprises a further miRNA or precursor thereof, wherein the further miRNA is selected from the group consisting of miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or an isomiR thereof, or a mimic thereof. It preferably further comprises an additional pharmaceutically active compound, preferably selected from the group consisting of a PP2A methylating agent, an inhibitor of hepatocyte growth factor (HGF), an antibody, a PI3K inhibitor, an Akt inhibitor, an mTOR inhibitor, a binder of a T cell co-stimulatory molecule such as a binder of OX40, and a chemotherapeutic agent.
The invention further provides a nanoparticle composition, for use as defined above, the nanoparticle comprising a diamino lipid and a miRNA-193a or a source thereof as defined above, wherein the diamino lipid is of general formula (I)
wherein

- n is 0, 1, or 2, and
- T¹, T², and T³are each independently a C₁₀-C₁₈chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.

Preferably the nanoparticles comprise 20-60 mol % of diamino lipid, and 0-40 mol % of a phospholipid, and 30-70 mol % of a sterol, and 0-10 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.
The invention also provides an in vivo, in vitro, or ex vivo method for agonising CRT, the method comprising the step of contacting a cell with a miRNA as defined above, or with a composition as defined above.
The invention also provides a method for treating a low-CRT cancer, the method comprising the step of administering to a subject a miRNA-193a as defined above, or a composition as defined above.

DESCRIPTION OF EMBODIMENTS

Surprisingly, the inventors identified miRNA-193a as a CRT agonist, allowing the use of miRNA-193a for treating diseases or conditions associated with low CRT expression, particularly low-CRT tumours. Accordingly, the invention provides a miRNA-193a or a source thereof, for use in treating a condition associated with low CRT expression. Such a miRNA-193a or a source thereof is referred to hereinafter as a miRNA for use according to the invention, or a miRNA-193a for use according to the invention. Preferably, the miRNA for use according to the invention is a CRT agonist. More preferably the miRNA for use according to the invention is for increasing or activating or inducing or promoting the calreticulin exposure pathway.
The miRNA for use according to the invention can also be for use in sensitizing tumor cells to PBMCs, preferably to T cells, which preferably comprises sensitizing tumor cells to PBMC ot T cell cytotoxicity. The miRNA for use according to the invention can also be for use in increasing the fulnerability of tumor cells to PBMCs, preferably to T cells, which preferably comprises increasing vulnerability of tumor cells to PBMC ot T cell cytotoxicity. Preferably the miRNA for use according to the invention is for stimulating release of signals from transfected tumor cells, wherein these signals can activate PBMCs, preferably T cells. Release of signals preferably comprises release of HGMB1, release of ATP, and/or surface expression of calreticulin (CRT), more preferably release of ATP and surface expression of CRT, most preferably surface expression of CRT.
As used herein, an “agonist of CRT” or “CRT agonist” refers to an agent that stimulates the production of CRT mRNA in a cell, or that stimulates the desired expression of CRT at a cell surface, or that repairs a defective pathway for surface display of CRT, or that stimulates expression of CRT protein in a cell, or stimulates the activity of CRT protein, or which can provide one or more of the functions of CRT, e.g., in regulating the expression of CRT on a cell surface. Preferably, a CRT agonist increases surface display or surface expression of CRT in cells, or restores the CRT surface expression pathway.
As used herein, low-CRT refers to cancers with a low level of CRT surface expression. CRT levels in for example the endoplasmatic reticulum can be normal, or also low, or even high. CRT surface expression is preferably determined according to the method described by Kuramitsu et al (Anticancer Res. 2010; 30:2093-2099). Briefly, the intensity of CRT staining in cancer cells from cancerous tissue is compared with that in the normal tissue. The percentage of positive cells is graded as 0, 0% immunopositive cells; 1, <50% positive cells; 2, ≥50% positive cells. And the staining intensity is graded as 0, negative; 1 weak; 2, moderate; 3, strong. The sum of the assigned values of the percentage of positive cells and the staining intensity is regarded as the immunoreactivity score. Scores between 0-2 are regarded as low-CRT, scores between 3-5 are regarded as high-CRT. Immunoreactivity for CRT expression is preferably evaluated by multiple different microscopists or by an automated image recognition tool.
miRNA, isomiR, Mimic, or a Source Thereof
MicroRNAs (miRNAs) are small RNAs of 17-25 nucleotides, which function as regulators of gene expression in eukaryotes. miRNAs are initially expressed in the nucleus as part of long primary transcripts called primary miRNAs (pri-miRNAs). Inside the nucleus, pri-miRNAs are partially digested by the enzyme Drosha, to form 65-120 nucleotide-long hairpin precursor miRNAs (pre-miRNAs) that are exported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs, which are the active molecules. In animals, these short RNAs comprise a 5′ proximal “seed” region (generally nucleotides 2 to 8) which appears to be the primary determinant of the pairing specificity of the miRNA to the 3′ untranslated region (3′-UTR) of a target mRNA.
Each of the definitions given below concerning a miRNA molecule, a miRNA mimic or a miRNA isomiR or a source of any of those is to be used for each of the identified miRNAs, molecules or mimics or isomiRs or sources thereof mentioned in this application: miRNA-193a, miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or isomiRs or mimics or sources thereof. Preferred mature sequences (SEQ ID NOs: 51-57), seed sequences (SEQ ID NOs: 17-50, where SEQ ID NOs: 17-23 are seed sequences for canonical miRNAs and SEQ ID NOs: 24-50 are seed sequences for isomiRs), isomiR sequences (SEQ ID NOs: 58-125), or source sequences (RNA precursor as SEQ ID NOs: 1-8, or DNA encoding a RNA precursor as SEQ ID NOs: 9-16) of said miRNA molecule or mimic or isomiR thereof respectively are identified in the sequence listing.
In the context of this invention, a miRNA-193a refers to a miRNA-193a molecule (that is to the canonical oligonucleotide) or to an isomiR thereof or to a mimic thereof. Preferably, miRNA-193a is a miRNA-193a-3p, more preferably a miRNA-193a-3p molecule, isomiR, or mimic thereof, and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NO: 22 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more. For a miRNA-193a molecules (that is for the canonical miRNA) the preferred seed sequence is SEQ ID NO: 22. For an isomiR of miRNA-193a a preferred seed sequence is SEQ ID NO: 22.
A preferred mimic of miRNA-193a has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NO: 22 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 56, 121, 122, or 219, preferably 56 or 219, more preferably 219, and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 131, 196, 197, 206, or 218, more preferably 218, and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A mimic is a molecule which has a similar or identical activity with a miRNA molecule. In this context a similar activity is given the same meaning as an acceptable level of an activity. A mimic is, in a functional determination, opposed to an antagomir. Preferred mimics are synthetic oligonucleotides, preferably comprising one or more nucleotide analogues such as locked nucleic acid monomers, and/or nucleotides comprising scaffold modifications and/or nucleotides comprising base modifications. A mimic can be a mimic for a miRNA or for an isomiR. Preferred mimics are mimics for a miRNA or for an isomiR. Preferred mimics are double stranded mimics.
Preferred mimics are double stranded oligonucleotides comprising a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). The canonical miRNA as it naturally occurs is defined herein as having an antisense sequence, because it is complementary to the sense sequence of naturally occurring targets. It follows that in a double stranded mimic as is a preferred mimic for use according to the invention, there are two strands, one of which is designated as a sense strand, and one of which is designated as an antisense strand. The antisense strand can have the same sequence as a miRNA, or as a precursor of a miRNA, or as an isomiR, or it can have the same sequence as a fragment thereof, or comprise the same sequence, or comprise the same sequence as a fragment thereof. The sense strand is at least partially reverse complementary to the antisense strand, to allow formation of the double stranded mimic. The sense strand is not necessarily biologically active per se, one of its important functions is to stabilize the antisense strand or to prevent its degradation or to facilitate its delivery. An examples of a sense strand for a mature miRNA is SEQ ID NO: 131. Examples of sense strands for isomiRs are SEQ ID NOs: 196 or 197.
A preferred mimic of miRNA-193a has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NO: 22 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 56, 121, 122, or 219, preferably 56, more preferably 219, and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 131, 196, 197, 206, or 218, more preferably 218, and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
In preferred embodiments an antisense strand comprises at least one modified nucleoside, preferably selected from the group consisting of a bridged nucleic acid nucleoside such as a locked nucleic acid (LNA) nucleoside, a 2′-O-alkylnucleoside such as a 2′-O-methylnucleoside, a 2′-fluoronucleoside, and a 2′-azidonucleoside, preferably a 2′-O-alkylnucleoside such as a 2′-O-methylnucleoside. It is preferred that such an at least one modified nucleoside replaces the first or the last RNA nucleoside, or replaces the second or second-to-last RNA nucleoside. In preferred embodiments at least two modified nucleosides replace the first two or the last two RNA nucleosides. More preferably both the first and the last RNA nucleosides are replaced, even more preferably both the first two and the last two. It is to be understood that the replacing modified nucleoside has the same pairing capacity as the nucleoside it replaces, preferably it has the same nucleobase. Preferably an antisense strand does not comprise modified nucleosides outside of the first two or the last two RNA nucleosides. In preferred embodiments, the last base of an antisense strand is a DNA nucleoside; more preferably the last two bases of an antisense strand are DNA nucleosides. Preferably the last one or two residues of an antisense strand form an overhang when the antisense strand forms a pair with the sense strand; more preferably the last two residues of an antisense strand form such an overhang. Preferably an antisense sense does not comprise DNA nucleosides outside of the last two nucleosides, or outside of an overhang. Preferably a sense strand comprises only RNA nucleosides.
In preferred embodiments a sense strand comprises at least one modified nucleoside, preferably selected from the group consisting of a bridged nucleic acid nucleoside such as a locked nucleic acid (LNA) nucleoside, a 2′-O-alkylnucleoside such as a 2′-O-methylnucleoside, a 2′-fluoronucleoside, and a 2′-azidonucleoside, preferably a 2′-O-alkylnucleoside such as a 2′-O-methylnucleoside. It is preferred that such an at least one modified nucleoside replaces the first or the last RNA nucleoside, or replaces the second or second-to-last RNA nucleoside. In preferred embodiments at least two modified nucleosides replace the first two or the last two RNA nucleosides. More preferably both the first and the last RNA nucleosides are replaced, even more preferably both the first two and the last two. It is to be understood that the replacing modified nucleoside has the same pairing capacity as the nucleoside it replaces, preferably it has the same nucleobase. Preferably a sense strand does not comprise modified nucleosides outside of the first two or the last two RNA nucleosides. In preferred embodiments, the 3′ prime end of the sense strand is elongated by a DNA nucleoside; more preferably the last two bases of a sense strand are DNA nucleosides, even more preferably the DNA nucleoside is deoxythymidine. Preferably the last one or two residues of a sense strand form an overhang when the sense strand forms a pair with the antisense strand; more preferably the last two residues of a sense strand form such an overhang. Preferably a sense strand does not comprise DNA nucleosides outside of the last two nucleosides, or outside of an overhang. In particularly preferred embodiments a mimic comprises an antisense strand that comprises only RNA nucleosides and a sense strand that comprises modifications as described above.
Preferably, the sense strand and the antisense strand do not fully overlap, having one, two, three, or four additional bases at their 3′-end, preferably having two additional bases at their 3′-end, forming a sticky end. Accordingly, in the corresponding antisense strand, the 3′-end one, two, three, or four bases preferably do not have a reverse complementary base in the sense strand, also forming a sticky end; more preferably the first two bases of a sense strand form a sticky end, not having complementary bases in the antisense strand. The sense strand is not necessarily biologically active, it serves primarily to increase the stability of the antisense strand. Examples of preferred sequences for sense/antisense pairs for mimics are SEQ ID NOs: 206 and 218 for sense strands, more preferably SEQ ID NO: 218 for sense strands, and SEQ ID NO: 219 for antisense strands. A preferred pair is SEQ ID NOs: 206 or 218 and SEQ ID NO: 219, more preferably SEQ ID NO: 218 and SEQ ID NO: 219.
In preferred embodiments, a mimic is a double stranded oligonucleotide comprising a sense strand and an antisense strand, wherein both strands have a length of 15 to 30 nucleotides, preferably of 17 to 27 nucleotides, wherein the antisense strand has 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NOs: 56, 121, or 122, wherein the sense strand optionally has 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NOs: 131, 196 or 197, preferably 131 or 196, wherein the sense strand and the antisense strand preferably can anneal to form said double stranded oligonucleotide, wherein optionally one or both ends of the oligonucleotide are sticky ends having an overlap of one, two, three, or four, preferably of two nucleotides, wherein the sense strand optionally comprises chemically modified nucleotides. Preferably, the two strands of a double stranded mimic have the same length, or differ by one, two, three, four, five, or six nucleotides in length.
Within the whole text of the application unless otherwise indicated, a miRNA may also be named a miRNA molecule, a miR, an isomiR, or a mimic, or a source or a precursor thereof. Each sequence identified herein may be identified as being SEQ ID NO as used in the text of the application or as corresponding SEQ ID NO in the sequence listing. A SEQ ID NO as identified in this application may refer to the base sequence of said miRNA, isomiR, mimic, or source thereof such as a precursor. For all SEQ ID NOs, a skilled person knows that some bases can be interchanged. For example, each instance of T can be individually substituted by U, and vice versa. An RNA sequence provided for a mature miRNA can for example be synthesized as a DNA oligonucleotide using DNA nucleotides instead of RNA nucleotides. In such a case, thymine bases can be used instead of uracil bases. Alternately, thymine bases on deoxyribose scaffolds can be used. A skilled person understands that the base pairing behaviour is more important than the exact sequence, and that T and U are generally interchangeable for such purposes. Accordingly, a mimic can be either a DNA or an RNA molecule, or a further modified oligonucleotide as defined later herein.
In the context of the invention, a miRNA molecule or a mimic or an isomiR may be a synthetic or natural or recombinant or mature or part of a mature miRNA or a human miRNA or derived from a human miRNA as further defined in the part dedicated to the general definitions. A human miRNA molecule is a miRNA molecule which is found in a human cell, tissue, organ or body fluids (i.e. endogenous human miRNA molecule). A human miRNA molecule may also be a human miRNA molecule derived from an endogenous human miRNA molecule by substitution, deletion and/or addition of a nucleotide. A miRNA molecule or a mimic or an isomiR may be a single stranded or double stranded RNA molecule.
Preferably a miRNA molecule or a mimic or an isomiR thereof is from 6 to 30 nucleotides in length, preferably 12 to 30 nucleotides in length, preferably 15 to 28 nucleotides in length, more preferably said molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
In a preferred embodiment, a miRNA molecule or a mimic or isomiR comprises at least 6 of the 7 nucleotides present in the seed sequence of said miRNA molecule or a mimic or isomiR thereof (SEQ ID NOs: 17-50). Preferably in this embodiment, a miRNA molecule or a mimic or isomiR is from 6 to 30 nucleotides in length and more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence of said miRNA molecule or mimic or isomiR. Even more preferably a miRNA molecule or a mimic or isomiR is from 15 to 28 nucleotides in length and more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence, even more preferably a miRNA molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
In this context, to comprise at least 6 of the 7 nucleotides present in a seed sequence is intended to refer to a continuous stretch of 7 nucleotides that differs from the seed sequence in at most one position. Alternately, this can refer to a continuous stretch of 6 nucleotides that differs from the seed sequence only through omission of a single nucleotide. Throughout the application, more preferred miRNA molecules, isomiRs, mimics, or precursors thereof comprise all 7 of the 7 nucleotides present in an indicated seed sequence, or in other words have 100% sequence identity with said seed sequences. Preferably, when comprised in a miRNA, isomiR, or mimic, a seed sequence starts at nucleotide number 1, 2, or 3, and ends at nucleotide number 7, 8, 9, 10, or 11; most preferably such a seed sequence starts at nucleotide number 2 and ends at nucleotide number 8.
The miRNA-193a for use according to the invention can be combined with a further miRNA selected from the group consisting of miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or an isomiR thereof, or a mimic thereof.
A preferred miRNA-323 is a miRNA-323-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 17 or 24-28 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-323 has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 17 or 24-28 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 51, 58-68, or 209 and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 126, 133-143, 201, or 208 and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred miRNA-342 is a miRNA-342-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 18 or 29-42 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-342 has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 18 or 29-42 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 52, 69-113, or 211 and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 127, 144-188, 202, or 210 and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred miRNA-520f is a miRNA-520f-3p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 19 or 43-44 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-520f has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 19 or 43-44 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 53, 114, 115, or 213 and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 128, 189, 190, 203, or 212, and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A further preferred miRNA-520f is a miRNA-520f-3p-i3 molecule or mimic thereof comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NO: 20 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-520f-3p-i3 has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NO: 20 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 54 or 215, and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 129, 204, or 214 and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred miRNA-3157 is a miRNA-3157-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 21 or 45-48 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-3157 has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 21 or 45-48 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 55, 116-120, or 217, and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 130, 191-195, 205, or 216, and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred miRNA-7 is a miRNA-7-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 23 or 50 and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
A preferred mimic of miRNA-7 has a sense strand and an antisense strand, wherein the antisense strand comprises at least 6 of the 7 nucleotides present in the seed sequence of SEQ ID NOs: 23 or 50 and wherein the antisense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, and wherein the antisense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 57, 123-125, or 221, and wherein the sense strand preferably has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 132, 198-200, 207, or 220, and wherein the sense strand preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
Preferably, a miRNA molecule, isomiR, or mimic thereof has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more, comprises at least 6 of the 7 nucleotides present in a given seed sequence of any one of SEQ ID NOs: 17-50 and has at least 70% identity over the whole mature sequence of any one of SEQ ID NOs: 51-125. Preferably, identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.
Alternatively, preferably, a miRNA molecule, isomiR, or mimic thereof has a length of not more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides, comprises at least 6 of the 7 nucleotides present in a given seed sequence of any one of SEQ ID NOs: 17-50 and has at least 70% identity over the whole mature sequence of any one of SEQ ID NOs: 51-125. Preferably, identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.
In another preferred embodiment, an isomiR of a miRNA molecule has at least 70% identity over the whole isomiR sequence of any one of SEQ ID NOs: 58-125. Preferably, identity is at least 75%, 80%, 85%, 90%, 95% or higher. Preferably in this embodiment, an isomiR of a miRNA molecule or a mimic thereof has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.
Accordingly a preferred miRNA-323 molecule, isomiR, or mimic thereof is a miRNA-323-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 17, 24-28 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 51, 58-68 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-323 molecule, isomiR, or mimic thereof is a miRNA-323-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 17, 24-28 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 51, 58-68 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-342 molecule, isomiR, or mimic thereof is a miRNA-342-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 18, 29-42 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 52, 69-113 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-520f molecule, isomiR, or mimic thereof is a miRNA-520f-3p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 19, 43-44 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 53, 114-115 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more. A further preferred miRNA 520f molecule, isomiR, or mimic thereof is a miRNA-520f-3p-i3 molecule or a mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NO: 20 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NO: 54 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-3157 molecule, isomiR, or mimic thereof is a miRNA-3157-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 21, 45-48 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 55, 116-120 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-193a molecule, isomiR, or mimic thereof is a miRNA-193a-3p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NO: 22 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 56, 121-122 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Accordingly a preferred miRNA-7 molecule, isomiR, or mimic thereof is a miRNA-7-5p molecule, isomiR, or mimic thereof and comprises at least 6 of the 7 nucleotides present in the seed sequence identified as SEQ ID NOs: 23 or 50 and/or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity over SEQ ID NOs: 57, 123-125 and/or has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides or more.
Another preferred miRNA molecule, isomiR, or mimic thereof has at least 60% identity with a seed sequence of any one of SEQ ID NOs: 17-50, or with a mature sequence of any one of SEQ ID NOs: 51-57, or with a precursor sequence of any one of SEQ ID NOs: 1-16, preferably of any one of SEQ ID NOs: 1-8, or with a DNA encoding an RNA precursor of any one of SEQ ID NOs: 9-16, or with an isomiR sequence of any one of SEQ ID NOs: 58-125. Identity may be at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. Identity is preferably assessed on the whole SEQ ID NO as identified in a given SEQ ID NO. However, identity may also be assessed on part of a given SEQ ID NO. Part may mean at least 50% of the length of the SEQ ID NO, at least 60%, 70%, 80%, 90% or 100%.
A precursor sequence may result in more than one isomiR sequences depending on the maturation process—see for example miRNA-323 (mature sequence SEQ ID NO: 51) where in certain tissues multiple isomiRs have been identified (SEQ ID NOs: 58-68). IsomiRs of a miRNA molecule stem from the same precursor, and conversely a precursor can lead to multiple miRNA molecules, one of which is referred to as the canonical miRNA (such as miRNA-323-5p, SEQ ID NO: 51) and others being referred to as isomiRs (such as the oligonucleotide represented by SEQ ID NOs: 58-68). The difference between a canonical miRNA and its isomiRs can be said lie only in their prevalence—generally, the most prevalent molecule is called the canonical miRNA, while the others are isomiRs. Dependent on the type, environment, position in its life cycle, or pathological state of a cell, individual isomiRs or miRNAs can be expressed at different levels; expression can even differ between population groups or gender (Loher et al., Oncotarget (2014) DOI: 10.18632/oncotarget.2405).
The chemical structure of the nucleotides of a miRNA molecule or mimics or sources thereof, or of a sense strand or an antisense strand in a mimic of a miRNA or of an isomiR, may be modified to increase stability, binding affinity and/or specificity. Said sense strand or antisense strand may comprise or consists of a RNA molecule or preferably a modified RNA molecule. A preferred modified RNA molecule comprises a modified sugar. One example of such modification is the introduction of a 2′-O-methyl or 2′-O-methoxyethyl group or 2′ fluoride group on the nucleic acid to improve nuclease resistance and binding affinity to RNA. Another example of such modification is the introduction of a methylene bridge connecting the 2′-0 atom and the 4′-C atom of the nucleic acid to lock the conformation (Locked Nucleic Acid (LNA)) to improve affinity towards complementary single-stranded RNA. A third example is the introduction of a phosphorothioate group as linker between nucleic acid in the RNA-strand to improve stability against a nuclease attack. A fourth modification is conjugation of a lipophilic moiety on the 3′ end of the molecule, such as cholesterol to improve stability and cellular delivery.
In a preferred embodiment, the first two bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications. In a preferred embodiment, the first two of the last four bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications. In a preferred embodiment, the first two bases and the first two of the last four bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications. In a preferred embodiment, the last two bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications. In a preferred embodiment, the first two and the last two bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications. In a preferred embodiment, the last two bases of a sense strand of a mimic are DNA bases. In a preferred embodiment, the first two bases and the first two of the last four bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications, and the last two bases of said sense strand are DNA bases. In a preferred embodiment, the first two bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications, and the last two bases of said sense strand are DNA bases. In a preferred embodiment, the first two of the last four bases of a sense strand of a mimic have modified sugars, preferably 2′-O-methyl modifications, and the last two bases of said sense strand are DNA bases.
A source of a miRNA molecule or a source of a mimic or an isomiR may be any molecule which is able to induce the production of a miRNA molecule or of a mimic or isomiR as identified herein and which preferably comprises a hairpin-like structure and/or a double stranded nucleic acid molecule. The presence of a hairpin-like structure may be assessed using the RNAshapes program (Steffen P. et al 2006) using sliding windows of 80, 100 and 120 nt or more. The hairpin-like structure is usually present in a natural or endogenous source of a miRNA molecule whereas a double-stranded nucleic acid molecule is usually present in a recombinant or synthetic source of a miRNA molecule or of an isomiR or mimic thereof.
A source of a miRNA molecule or of a mimic or an isomiR may be a single stranded, a double stranded RNA or a partially double stranded RNA or may comprise three strands, an example of which is described in WO2008/10558. As used herein partially double stranded refers to double stranded structures that also comprise single stranded structures at the 5′ and/or at the 3′ end. It may occur when each strand of a miRNA molecule does not have the same length. In general, such partial double stranded miRNA molecule may have less than 75% double stranded structure and more than 25% single stranded structure, or less than 50% double stranded structure and more than 50% single stranded structure, or more preferably less than 25%, 20% or 15% double stranded structure and more than 75%, 80%, 85% single stranded structure.
Alternatively, a source of a miRNA molecule or of a mimic or an isomiR thereof is a DNA molecule encoding a precursor of a miRNA molecule or a mimic or an isomiR thereof. Preferred DNA molecules in this context are SEQ ID NOs: 9-16. For the miRNA for use according to the invention, SEQ ID NO: 13 is preferred. The invention encompasses the use of a DNA molecule encoding a precursor of a miRNA molecule that has at least 70% identity with said SEQ ID NO: 13. Preferably, the identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. Preferably in this embodiment, a DNA molecule has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and has at least 70% identity with a DNA sequence of SEQ ID NOs: 13.
The induction of the production of a given miRNA molecule or of a mimic or an isomiR is preferably obtained when said source is introduced into a cell using one assay as defined below. Cells encompassed by the present invention are later on defined.
A preferred source of a miRNA molecule or of a mimic or an isomiR thereof is a precursor thereof, more preferably a nucleic acid encoding said miRNA molecule or a mimic or an isomiR thereof. A preferred precursor is a naturally-occurring precursor. A precursor may be a synthetic or recombinant precursor. A synthetic or recombinant precursor may be a vector that can express a naturally-occurring precursor. In preferred embodiments, this aspect provides the miRNA for use according to the invention, wherein a source of a miRNA is a precursor of a miRNA and is a nucleic acid of at least 50 nucleotides in length. In preferred embodiments is provided the miRNA-193a or a source thereof for use according to the invention, wherein said miRNA shares at least 70% sequence identity with any one of SEQ ID NOs: 56, 121, or 122, and/or wherein said miRNA is from 15-30 nucleotides in length, and/or wherein said source of a miRNA is a precursor of said miRNA and shares at least 70% sequence identity with any one of SEQ ID NOs: 5 or 13. More preferably the miRNA-193a for use according to the invention shares at least 70% sequence identity with any one of SEQ ID NOs: 56, 121, or 122, and is from 15-30 nucleotides in length; more preferably said source of a miRNA-193a is a precursor of said miRNA-193a and shares at least 70% sequence identity with any one of SEQ ID NOs: 5 or 13.
A preferred precursor of a given miRNA molecule has a sequence represented by any one of SEQ ID NOs: 1-16. The invention encompasses the use of a precursor of a miRNA molecule or of an isomiR or mimic thereof that has at least 70% identity with said sequence. Preferably, identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. Preferably in this embodiment, a DNA molecule has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and has at least 70% identity with a sequence represented by any one of SEQ ID NOs: 1-16. Preferably, in this embodiment, a precursor comprises a seed sequence that shares at least 6 of the 7 nucleotides with a seed sequence selected from the group represented by SEQ ID NOs: 17-50. More preferably, a precursor comprises a seed sequence selected from the group represented by SEQ ID NOs: 17-50. A more preferred precursor of a given miRNA molecule has a sequence represented by any one of SEQ ID NOs: 1-8. The invention encompasses the use of a precursor of a miRNA molecule or of an isomiR or mimic thereof that has at least 70% identity with said sequence. Preferably, identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. Preferably in this embodiment, a DNA molecule has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and has at least 70% identity with a sequence represented by any one of SEQ ID NOs: 1-8. Preferably, in this embodiment, a precursor comprises a seed sequence that shares at least 6 of the 7 nucleotides with a seed sequence selected from the group represented by SEQ ID NOs: 17-50. More preferably, a precursor comprises a seed sequence selected from the group represented by SEQ ID NOs: 17-50.
Accordingly, a preferred source of a miRNA-323 molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 1 or 9, preferably SEQ ID NO: 1, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NOs: 17 or 24-28. Such a source is a precursor of a miRNA-323 molecule and of miRNA-323 isomiRs.
Accordingly, a preferred source of a miRNA-342 molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 2 or 10, preferably SEQ ID NO: 2, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NOs: 18 or 29-42. Such a source is a precursor of a miRNA-342 molecule and of miRNA-342 isomiRs.
Accordingly, a preferred source of a miRNA-520f molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 3 or 11, preferably SEQ ID NO: 3, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NOs: 19, 20, 43, or 44. Such a source is a precursor of a miRNA-520f molecule and of miRNA-520f isomiRs such as miRNA-520f-3p-i3.
Accordingly, a preferred source of a miRNA-3157 molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 4 or 12, preferably SEQ ID NO: 4, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NOs: 21 or 45-48. Such a source is a precursor of a miRNA-3157 molecule and of miRNA-3157 isomiRs.
Accordingly, a preferred source of a miRNA-193a molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 5 or 13, preferably SEQ ID NO: 5, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NO: 22. Such a source is a precursor of a miRNA-193a molecule and of miRNA-193a isomiRs.
Accordingly, a preferred source of a miRNA-7 molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NOs: 6-8 or 14-16, preferably SEQ ID NOs: 6-8, and optionally has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and optionally comprises a seed sequence that shares at least 6 of the 7 nucleotides of any one of SEQ ID NOs: 23 or 50. Such a source is a precursor of a miRNA-7 molecule and of miRNA-7 isomiRs.
In this context, it is pointed that several precursors of a given mature miRNA molecule may lead to an identical miRNA molecule. For example, miRNA-7 may originate from precursor miRNA-7-1 or miRNA-7-2 or miRNA-7-3 (preferably identified as being SEQ ID NOs: 6, 8, or 8, respectively). Also in this context, it is pointed that several isomirs of a given mature miRNA molecule may lead to miRNA molecules with identical seed sequences. For example, mature miRNA-323-5p (SEQ ID NO: 51) and at least isomirs with SEQ ID NOs: 58 or 59 all share the same seed sequence (preferably identified as being SEQ ID NO: 17).
Preferred sources or precursors have been defined elsewhere herein. A preferred source includes or comprises an expression construct comprising a nucleic acid, i.e. DNA encoding said precursor of said miRNA, more preferably said expression construct is a viral gene therapy vector selected from gene therapy vectors based on an adenovirus, an adeno-associated virus (AAV), a herpes virus, a pox virus and a retrovirus. A preferred viral gene therapy vector is an AAV or Lentiviral vector. Other preferred vectors are oncolytic viral vectors. Such vectors are further described herein below. Alternatively, a source may be a synthetic miRNA molecule or a chemical mimic as further defined in the part dedicated to general definitions.
Conditions Associated with PTEN-Deficiency
The use according to the invention is use in treating a condition associated with PTEN-deficiency. Such a condition, or disease, is referred to herein as a PTEN-deficient condition. The invention provides this new medical use of miRNA-193a. This use can also be the use of the composition or miRNA in the manufacture of a medicament. Compositions are defined in a later section. Treatment preferably refers to preventing, ameliorating, reverting, curing and/or delaying a condition. When the PTEN-deficient condition is a PTEN-deficient tumour, preferred treatment can be obtaining an anti-tumour effect.
By the term “treating” and derivatives thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition; (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition; (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or one or more of the symptoms, effects or side effects associated with the condition or treatment thereof; (4) to slow the progression of the condition or one or more of the biological manifestations of the condition and/or (5) to cure said condition or one or more of the biological manifestations of the condition by eliminating or reducing (preferably to undetectable levels) one or more of the biological manifestations of the condition for a period of time considered to be a state of remission for that manifestation without additional treatment over the period of remission. One skilled in the art will understand the duration of time considered to be remission for a particular disease or condition. Prophylactic therapy is also contemplated. The skilled artisan will appreciate that “prevention” is not always an absolute term. In medicine, “prevention” is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen, or when PTEN-deficiency is diagnosed in a patient.
T cell-mediated immunotherapies are promising cancer treatments. However, many patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. Loss of PTEN in tumour cells in preclinical models of melanoma inhibits T cell-mediated tumour killing and decreases T cell trafficking into tumours. In patients (e.g., subjects), PTEN loss correlates with decreased T cell infiltration at tumour sites, reduced likelihood of successful T cell expansion from resected tumours, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumour cells increased the expression of immunosuppressive cytokines, resulting in decreased T cell infiltration in tumours, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective RI3Kb (PI3Kb) inhibitor can improve the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. These findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. See Peng et al., Cancer Discovery 6:202-216 (2016).
The PI3K pathway plays a critical role in cancer by regulating several critical cellular processes, including proliferation and survival. One of the most common ways that this pathway is activated in cancer is by loss of expression of the tumour suppressor PTEN, which is a lipid phosphatase that dampens the activity of PI3K signalling. Loss of PTEN corresponds with increased activation of the PI3K-AKT pathway in multiple tumour types. Loss of PTEN is not universal in cancer—for example, it occurs in up to 30% of melanomas.
As used herein, “PTEN deficient” or “PTEN deficiency” preferably refers to a condition caused by or exacerbated by a deficiency of the tumour suppressor function of PTEN, e.g., loss of expression of the PTEN tumour suppressor. Such deficiency preferably includes mutation in the PTEN gene, reduction or absence of PTEN protein when compared to PTEN wild-type, or mutation or absence of other genes that cause suppression of PTEN function. It more preferably includes PTEN activity or expression lost by deletion, mutation, or through epigenetic changes. Multiple mechanisms exist for the regulation of PTEN, including transcription, mRNA stability, miRNA targeting, translation, and protein stability. PTEN is transcriptionally silenced by promoter methylation in PTEN-deficient endometrial, gastric, lung, thyroid, breast and ovarian tumours, as well as glioblastoma. Mutations resulting in the loss of function or reduced levels of PTEN, as well as PTEN deletions or alteration are found in several sporadic tumours. See Aguissa-Toure et al., Cellular and Molecular Life Sciences 69: 1475-1491 (2012). A skilled person knows how to determine whether a condition such as a cancer is PTEN deficient. PTEN deficiency can be determined by methods such as Q-PCR or ELISA or immunohistochemistry. Human PTEN qPCR primer pairs are commercially available, e.g., from Sino Biological and Genecopoeia. A PTEN (Human) ELISA kit is commercially available, e.g., from BioVision and Abeam. An immunohistochemistry protocol is provided, e.g., in Sakr et al., Appl. Immunohistochem. Mol. Morphol. 18:371-374 (2010). PTEN antibodies are commercially available, e.g., from Abeam and Sino Biological. For reference, the human PTEN mRNA sequence is NCBI Accession No. NM_000314.4; the protein sequence is NCBI Accession No. AAH05821.1.
PTEN-deficient conditions are known in the art, and as described above the PTEN-deficient nature of a condition can be readily established using routine assays. Examples of conditions of which PTEN-deficient variants exist are cancer, autism, macrocephaly, benign tumours, and non-cancerous neoplasia. Preferred conditions of which PTEN-deficient variants exist are cancer, benign tumours, and non-cancerous neoplasia, which are herein collectively referred to as PTEN-deficient tumours. Examples of non-cancerous neoplasia are hamartoma such as those occurring in Bannayan-Zonana syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Proteus-like syndrome, Cowden disease, PTEN hamartoma tumour syndrome (PHTS), and Lhermitte-Duclos disease. A most highly preferred PTEN-deficient tumour is a PTEN-deficient cancer.
A preferred PTEN-deficient condition is a tumour, in other words a preferred use according to the invention is in treating a PTEN-deficient tumour, more preferably a PTEN-deficient cancer. Generally, as used herein, reference to treatment of cancer is intended to refer to treatment of PTEN-deficient cancer. Unless otherwise indicated, an anti-tumour effect is preferably assessed or detected before treatment and after at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or more in a treated subject. An anti-tumour effect is preferably identified in a subject as:

- an inhibition of proliferation or a detectable decrease of proliferation of tumour cells or a decrease in cell viability of tumour cells or melanocytes, and/or
- an increase in the capacity of differentiation of tumour cells, and/or
- an increase in tumour cell death, which is equivalent to a decrease in tumour cell survival, and/or
- a delay in occurrence of metastases and/or of tumour cell migration, and/or
- an inhibition or prevention or delay of the increase of a tumour weight or growth, and/or
- a prolongation of patient survival of at least one month, several months or more (compared to those not treated or treated with a control or compared with the subject at the onset of the treatment), and/or
- a decrease in tumour size or volume.

In the context of the invention, a patient may survive and may be considered as being disease free. Alternatively, the disease or condition may have been stopped or delayed or regressed. An inhibition of the proliferation of tumour cells may be at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Proliferation of cells may be assessed using known techniques. An decrease in cell viability of tumour cells or melanocytes may be a decrease of at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Such decrease may be assessed 4 days after transfection with a given miRNA molecule, equivalent or source thereof. Cell viability may be assessed via known techniques such as the MTS assay.
Treatment of tumour or cancer can be the reduction of tumour volume or a decrease of tumour cell viability. Reduction of tumour volume can be assessed using a calliper. A decrease of tumour volume or cell viability or survival may be at least a decrease of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. An induction of apoptosis in tumour cells or an induction of tumour cell death may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumour cell viability or survival or death may be assessed using techniques known to the skilled person. Tumour cell viability and death may be assessed using routine imaging methods such as MRI, CT or PET, and derivatives thereof, or in biopsies. Tumour cell viability may be assessed by visualising the extension of the lesion at several time points. A decrease of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more of the lesion observed at least once will be seen as a decrease of tumour cell viability.
An inhibition of the proliferation of tumour cells may be at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Proliferation of cells may be assessed using known techniques as a standard proliferation assay. Such a proliferation assay may use of vital stains such as Cell Titer Blue (Promega). This includes a substrate molecule that is converted into a fluorescent molecule by metabolic enzymes. The level of fluorescence then reflects the number of living and metabolically active cells. Alternatively, such proliferation assay may determine the mitotic index. The mitotic index is based on the number of tumour cells under proliferation stage compared to the number of total tumour cells. The labelling of proliferative cells can be performed by using the antibody Ki-67 and immunohistochemistry staining. An inhibition of the proliferation of tumours cells may be seen when the mitotic index is reduced by at least 20%, at least 30%, at least 50% or more (as described in Kearsley J. H., et al, 1990, PMID: 2372483).
A delay in occurrence of metastases and/or of tumour cell migration may be a delay of at least one week, one month, several months, one year or longer. The presence of metastases may be assessed using MRI, CT or Echography or techniques allowing the detection of circulating tumour cells (CTC). Examples of the latter tests are CellSearch CTC test (Veridex), an EpCam-based magnetic sorting of CTCs from peripheral blood.
In certain embodiments, an inhibition or a decrease of a tumour weight or a delayed tumour growth or an inhibition of a tumour growth may be of at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumour weight or volume tumour growth may be assessed using techniques known to the skilled person. The detection of tumour growth or the detection of the proliferation of tumour cells may be assessed in vivo by measuring changes in glucose utilization by positron emission tomography with the glucose analogue 2-[¹⁸F]-fluor-2-deoxy-D-glucose (FDG-PET) or [¹⁸F]-3′-fluoro-3′-deoxy-L-thymidine PET. An ex vivo alternative may be staining of a tumour biopsy with Ki67. An increase in the capacity of differentiation of tumour cells may be assessed using a specific differentiation marker and following the presence of such marker on cells treated. Preferred markers or parameters are p16, Trp-1 and PLZF, c-Kit, MITF, Tyrosinase, and Melanin. This may be done using RT-PCR, western blotting or immunohistochemistry. An increase of the capacity of differentiation may be at least a detectable increase after at least one week of treatment using any of the identified techniques. Preferably, the increase is of 1%, 5%, 10%, 15%, 20%, 25%, or more, which means that the number of differentiated cells within a given sample will increase accordingly. In certain embodiments, tumour growth may be delayed at least one week, one month, two months or more. In a certain embodiment, an occurrence of metastases is delayed at least one week, two weeks, three weeks, four weeks, one months, two months, three months, four months, five months, six months or more.
In preferred embodiments, the PTEN-deficient tumour is a PTEN-deficient sarcoma, brain cancer, head and neck cancer, breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, ovarian cancer, melanoma, pancreatic cancer, thyroid cancer, hamartoma, tumour of the haematopoietic and lymphoid malignancy, or prostate cancer. In other more preferred embodiments, the PTEN-deficient tumour is a PTEN-deficient sarcoma, brain cancer, head and neck cancer, breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, ovarian cancer, pancreatic cancer, thyroid cancer, hamartoma, tumour of the haematopoietic and lymphoid malignancy, or prostate cancer. In other more preferred embodiments, the PTEN-deficient tumour is a PTEN-deficient sarcoma, brain cancer, head and neck cancer, ovarian cancer, thyroid cancer, or hamartoma. In other more preferred embodiments, the PTEN-deficient tumour is a PTEN-deficient lung cancer (preferably non small cell lung cancer), liver cancer (preferably hepatocellular carcinoma), breast cancer (preferably triple-negative breast cancer), and melanoma (preferably melanoma with an activating BRAF mutation). In other more preferred embodiments, the PTEN-deficient tumour is a PTEN-deficient lung cancer (preferably non small cell lung cancer), liver cancer (preferably hepatocellular carcinoma), or breast cancer (preferably triple-negative breast cancer).
Further examples of cancers that are suitable for treatment according to the invention include, but are not limited to, both primary and metastatic forms of head and neck, breast, lung, colon, ovary, and prostate cancers. Preferably the cancer is selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumour, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumour of bone, thyroid, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Erythroleukemia, malignant lymphoma, hodgkins lymphoma, non-hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumour) and testicular cancer. Preferred hamartoma are Bannayan-Zonana syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Proteus-like syndrome, Cowden disease, PTEN hamartoma tumour syndrome (PHTS), and Lhermitte-Duclos disease.
Additionally, examples of a cancer to be treated (when PTEN-deficient) include Barret's adenocarcinoma; billiary tract carcinomas; breast cancer; cervical cancer; cholangiocarcinoma; central nervous system tumours including primary CNS tumours such as glioblastomas, astrocytomas (e.g., glioblastoma multiforme) and ependymomas, and secondary CNS tumours (i.e., metastases to the central nervous system of tumours originating outside of the central nervous system); colorectal cancer including large intestinal colon carcinoma; gastric cancer; carcinoma of the head and neck including squamous cell carcinoma of the head and neck; hematologic cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplastic syndromes, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, megakaryoblastic leukemia, multiple myeloma and erythroleukemia; hepatocellular carcinoma; lung cancer including small cell lung cancer and non-small cell lung cancer; ovarian cancer; endometrial cancer; pancreatic cancer; pituitary adenoma; prostate cancer; renal cancer; sarcoma; skin cancers including melanomas; and thyroid cancers.
In preferred embodiments the cancer is selected from the group consisting of: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and thyroid cancer. In other preferred embodiments the cancer is selected from the group consisting of: ovarian, breast cancer, pancreatic cancer and prostate cancer. In other preferred embodiments the cancer is non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer or metastatic hormone-refractory prostate cancer. In other preferred embodiments the cancer is breast cancer, thyroid cancer, glioblastoma, endometrial cancer, prostate cancer, or melanoma. In other preferred embodiments the cancer is breast cancer, thyroid cancer, glioblastoma, endometrial cancer, or prostate cancer.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant cancer such as sorafenib-resistant cancer.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of carcinoma. More perferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant carcinoma such as sorafenib-resistant carcinoma.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of hepatocellular carcinoma (HCC). More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant HCC such as hepatocellular carcinoma (HCC) that is resistant to receptor tyrosine kinase inhibitors such as VEGF receptor inhibitors, for example axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, or vandetanib, preferably sorafenib.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of non-small-cell lung carcinoma (NSCLC). More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant NSCLC such as NSCLC that is resistant to platinum-based cell-cycle nonspecific antineoplastic agents (for example carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, or satraplatin, preferably cisplatin or carboplatin), or that is resistant to taxanes (for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel or docetaxel, more preferably paclitaxel), or that is resistant to pyrimidine-based antimetabolites (for example fluorouracil, capecitabine, doxifluridine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, or decitabine, preferably gemcitabine), or that is resistant to vinca alkaloids (for example vinblastine, vincristine, vinflunine, vindesine, or vinorelbine, preferably vinorelbine), or that is resistant to folic acid antimetabolites (aminopterin, methotrexate, pemetrexed, pralatrexate, or raltitrexed, preferably pemetrexed).
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of triple-negative breast cancer (TNBC). More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant TNBC such as anthracyclin-resistant TNBC, for example TNBC resistant to aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin, preferably to doxorubicin.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of melanoma. More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant melanoma such as melanoma that is resistant to nonclassical cell-cycle nonspecific antineoplastic agents (for example procarbazine, dacarbazine, temozolomide, altretamine, mitobronitol, or pipobroman, preferably dacarbazine or temozolomide), or that is resitant to taxanes (for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel such as albumin-bound paclitaxel), or that is resistant to platinum-based cell-cycle nonspecific antineoplastic agents (for example carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, or satraplatin, preferably cisplatin or carboplatin), or that is resistant to vinca alkaloids (for example vinblastine, vincristine, vinflunine, vindesine, or vinorelbine, preferably vinblastine). In other preferred embodiments, the miRNA for use according to the invention is not for use in the treatment of melanoma.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of pancreas cancer. More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant pancreas cancer such as pancreas cancer that is resitant to taxanes (for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel such as albumin-bound paclitaxel), or that is resistant to pyrimidine-based antimetabolites (for example fluorouracil, capecitabine, doxifluridine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, or decitabine, preferably fluorouracil or gemcitabine), or that is resistant to topoisomerase inhibitors (for example camptothecin, cositecan, belotecan, gimatecan, exatecan irinotecan, lurtotecan, silatecan, topotecan, rubitecan, preferably irinotecan).
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of colon cancer. More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant colon cancer such as colon cancer that is resistant to pyrimidine-based antimetabolites (for example fluorouracil, capecitabine, doxifluridine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, or decitabine, preferably fluorouracil or capecitabine), or that is resistant to topoisomerase inhibitors (for example camptothecin, cositecan, belotecan, gimatecan, exatecan irinotecan, lurtotecan, silatecan, topotecan, rubitecan, preferably irinotecan), or that is resistant to platinum-based cell-cycle nonspecific antineoplastic agents (for example carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, or satraplatin, preferably oxaliplatin), or that is resistant to trifluridine or tipiracil, or a combination of trifluridine and tipiracil.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of renal cell cancer (RCC). More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant RCC such as RCC that is resistant to receptor tyrosine kinase inhibitors such as VEGF receptor inhibitors, for example axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, or vandetanib, preferably suntinib, sorafenib, or pazopanib, more preferably sorafenib.
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of head and neck cancer (HNSCC). More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant HNSCC such as HNSCC that is resistant to taxanes (for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel or docetaxel), or that is resistant to pyrimidine-based antimetabolites (for example fluorouracil, capecitabine, doxifluridine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, or decitabine, preferably fluorouracil), or that is resistant to folic acid antimetabolites (aminopterin, methotrexate, pemetrexed, pralatrexate, or raltitrexed, preferably methotrexate), or that is resistant to platinum-based cell-cycle nonspecific antineoplastic agents (for example carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, or satraplatin, preferably cisplatin), or that is resistant to anthracyclins (for example aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin, preferably doxorubicin), or that is resistant to intercalating crosslinking agents (for example actinomycin, bleomycin, mitomycins, plicamycin, preferably bleomycin or mitomycin).
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of prostate cancer. More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant prostate cancer such as prostate cancer that is resistant to taxanes (for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably docetaxel), or that is resistant to anthracenediones (for example mitoxantrone or pixantrone, preferably mitoxantrone), or that is resistant to alkylating antineoplastic agents (for example estrogen-based alkylating antineoplastic agents such as alestramustine, atrimustine, cytestrol acetate, estradiol mustard, estramustine, estromustine, stilbostat; or phenestrol, preferably estramustine).
In preferred embodiments, the miRNA for use according to the invention is for use in the treatment of tumours of the haematopoietic and lymphoid malignancies. More preferably, the miRNA for use according to the invention is for use in the treatment of chemotherapy-resistant tumours of the haematopoietic and lymphoid malignancies such as myeloma that is resistant to bortezomib, or that is resistant to lenalidomide, or such as lymphoma that is resistant to CHOP or to rituximab, such as resistance to cyclophosphamide or to anthracyclines such as hydroxydaunorubicin or to oncovin or to prednisone, or such as leukemia resistant to vincristine, anthracyclines such as doxorubicine, L-asparaginase, cyclophosphamide, methotrexate, 6-mercaptopurine, chlorambucil, cyclophosphamide, corticosteroids such as prednisone or prednisolone, fludarabine, pentostatin, or cladribine. Treatment of chemotherapy-resistant cancer such as sorafenib-resitant cancer as described herein can be as second line treatment when chemotherapy such as sorafenib treatment has been found to be ineffective, or to be less effective than anticipated or desired.
Solid tumours are often epithelial in origin (i.e. carcinomas). A loss of epithelial cell markers (e.g. E-cadherin) and gain of mesenchymal cell markers (e.g. N-cadherin and Vimentin) is known for patient tumour samples, including prostate cancer. Cancer cells can dedifferentiate through this so-called Epithelial to Mesenchymal Transition (EMT). During EMT, intercellular cell junctions are broken down, thereby giving tumour cells the ability to migrate and invade into the surrounding tissue or through blood vessel walls. Such phenotypic changes play a major role in dissemination of the disease and ultimately lead to disease progression, which is often associated with poor prognosis for the patients.
Loss of E-cadherin expression is considered as a molecular hallmark of EMT. EMT in tumour cells results from a transcriptional reprogramming of the cell. In particular the transcriptional repression of the E-cadherin (CDH1) gene promoter has been shown to trigger the EMT phenotype. The E-cadherin protein is one of the most important cadherin molecules mediating cell-cell contacts in epithelial cells/tissues. CDH1 is repressed by binding of the transcriptional repressors, SNAI1, SNAI2, TCF3, TWIST, ZEB1, ZEB2 or KLF8, to three so-called E-boxes in the CDH1 proximal promoter region. Inhibiting the binding of these repressors to the CDH1 promoter can revert EMT, also called mesenchymal to epithelial transition (MET), and inhibits tumour cell invasion and tumour progression.
In preferred embodiments, the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of a disease or a condition associated with EMT, when such a disease or condition is associated with PTEN-deficiency. Herein the miRNA is preferably combined with a miRNA-518b molecule, miRNA-520f molecule, or a miRNA-524 molecule; or an isomiR or mimic thereof, or a precursor thereof. The disease or condition associated with EMT is preferably a cancer, more preferably a bladder or prostate cancer. This use is preferably by inducing a mesenchymal to epithelial transition.
In preferred embodiments, the composition for use according to the invention (compositions for use according to the invention are defined later herein) or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by downregulating the immunosuppressive tumour microenvironment. In related preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by preventing or reducing evasion of host immunity by a tumour. Such use is preferably for preventing, inhibiting, or reducing adenosine generation, for example by inhibiting or reducing activity of cell surface ectoenzymes such as those that dephosphorylate ATP to produce adenosine. Such use is more preferably for reducing NT5E expression and/or reducing ENTPD1 expression and/or inhibiting adenosine generation. More preferably, the composition for use according to the invention or the miRNA for use according to the invention is for reducing NT5E expression. More preferably, this composition for use according to the invention or this miRNA for use according to the invention is for reducing ENTPD1 expression. More preferably, this composition for use according to the invention or this miRNA for use according to the invention is for inhibiting adenosine generation. In even more preferred embodiments, this composition for use according to the invention or this miRNA for use according to the invention is for reducing cancer cell migration, preferably for reducing adenosine-induced cancer cell migration, most preferably for reducing adenosine-induced cancer cell migration associated with NT5E expression. Reduction of NT5E or ENTPD1 expression is preferably assessed by luciferase assay or by RT-PCR. Reduction of cancer cell migration is preferably assessed by in vitro transwell assays.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by promoting or increasing G2/M arrest in cancer cells, preferably in liver cancer cells, in lung cancer cells, in pancreatic cancer cells, in carcinoma cells, or in melanoma cells, more preferably in liver cancer cells, in carcinoma cells, or in melanoma cells, even more preferably in hepatocellular carcinoma cells or in melanoma cells. Such use is preferably for reducing the expression or activity of factors that regulate cell division and/or proliferation by associating with the cytoskeleton, such as MPP2 and/or STMN1. Such use is preferably for promoting or increasing factors that bind and/or sequester cyclin-dependent kinases, such as YWHAZ and/or CCNA2. Preferably, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by reducing the expression or activity of at least one of MPP2, STMN1, YWHAZ, and CCNA2, more preferably by reducing the expression or activity of at least YWHAZ or STMN1, even more preferably of at least YWHAZ, most preferably of each of MPP2, STMN1, YWHAZ, and CCNA2. Increase in G2/M arrest is preferably an increase as compared to untreated cells, and is preferably an increase of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more. It is preferably assessed by DNA staining followed by microscopy imaging to determine nucleus intensity based on DNA content. Reduction of the expression or activity of at least one of MPP2, STMN1, YWHAZ, and CCNA2 is preferably assessed using RT-PCR.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or reducing cancer cell migration, cancer cell adhesion, or cancer cell proliferation, or by increasing or promoting cancer cell apoptosis. These cancer cells are preferably lung cancer cells, liver cancer cells, breast cancer cells, melanoma cells, or carcinoma cells, more preferably lung cancer cells, liver cancer cells, breast cancer cells, or melanoma cells, even more preferably lung cancer cells such as A549 and H460, liver cancer cells such as Hep3B and Huh7, breast cancer cells such as BT549, and skin cancer cells such as A2058. In more preferred embodiments this use in treatment, prevention, delay, or amelioration of cancer is by decreasing expression or activity of at least one gene selected from the group consisting of FOXRED2, ERMP1, NTSE, SHMT2, HYOU1, TWISTNB, AP2M1, CLSTN1, TNFRSF21, DAZAP2, C1QBP, STARD7, ATPSSL, DCAF7, DHCR24, DPY19L1, AGPAT1, SLC30A7, AIMP2, UBP1, RUSC1, DCTNS, ATP5F1, CCDC28A, SLC35D2, WSB2, SEC61A1, MPP2, FAM60A, PITPNB, and POLE3, even more preferably from the group consisting of NTSE and TNFRSF21; preferably the use as described above for apoptosis, cell migration, adhesion, and proliferation is use for apoptosis, cell migration, adhesion, and/or proliferation associated with at least one of these genes. Expression is preferably assessed by RT-PCR.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by increasing or promoting apoptosis of cancer cells, preferably by increasing or promoting apoptosis associated with at least one gene selected from the group consisting of KCNMA1, NOTCH2, TNFRSF21, YWHAZ, CADM1, NOTCH1, CRYAA, ETS1, AIMP2, SQSTM1, ZMAT3, TGM2, CECR2, PDE3A, STRADB, NIPA1, MAPK8, TP53INP1, PRNP, PRT1, GCH1, DHCR24, TGFB2, NET1, PHLDA2, and TPP1, more preferably from the group consisting of NOTCH2, TNFRSF21, YWHAZ, ETS1, TGFB2, and MAPK8. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting angiogenesis, preferably angiogenesis associated with cancer cells, more preferably by decreasing or inhibiting angiogenesis associated with at least one gene selected from the group consisting of CRKL, CTGF, ZMIZ1, TGM2, ELK3, LOX, UBP1, PLAU, CYR61, and TGFB2, even more preferably CRKL, TGFB2 or PLAU, most preferably PLAU. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by modulating the unfolded protein response in cancer cells, more preferably by modulating the unfolded protein response associated with at least one gene selected from the group consisting of ERMP1, NCEH1, SEC31A, CLSTN1, FOXRED2, SEPN1, EXTL2, HYOU1, SLC35D1, SULF2, PTPLB, HHAT, ERAP2, FAF2, DPM3, PDZD2, SEC61A1, DHCR24, IDS, MOSPD2, DPM, PRNP, and AGPAT1. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention. Modulation of the unfolded protein response is preferably an inhibition or reduction of the unfolded protein response.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting chemotaxis of cancer cells, more preferably by decreasing or inhibiting chemotaxis associated with at least one gene selected from the group consisting of CXCL1, RAC2, CXCL5, CYR61, PLAUR, KCNMA1, AB12, and HPRT1, most preferably PLAUR. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting protein transport in cancer cells, more preferably by decreasing or inhibiting protein transport associated with at least one gene selected from the group consisting of STON2, RAB11FIP5, SRP54, YWHAZ, SYNRG, GCH1, THBS4, SRP54, TOMM20, SEC31A, TPP1, SLC30A7, TGFB2, AKAP12, AP2M1, ITGB3, GNAI3, SORL1, KRAS, SLC15A1, SEC61A1, APPL1, LRP4, PLEKHA8, STRADB, SCAMP4, HFE, CADM1, ZMAT3, ARF3, VAMPS, NUP50, DHCR24, RAB11FIP5, ATP6V1B2, SQSTM1, and WNK4, even more preferably YWHAZ, TGFB2, or KRAS, most preferably YWHAZ. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting nucleoside metabolism in cancer cells, more preferably by decreasing or inhibiting nucleoside metabolism associated with at least one gene selected from the group consisting of NUDT3, NUDT15, NUDT21, DERA, NT5E, GCH1, and HPRT1, most preferably NT5E. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting glycosylation of cancer cells, more preferably by decreasing or inhibiting glycosylation associated with at least one gene selected from the group consisting of SLC35D1, ST3GAL5, SULF2, LAT2, GALNT1, NCEH1, ST3GAL4, CHST14, B3GNT3, DPM3, GALNT13, DHCR24, NUDT15, IDH2, PPTC7, HPRT1, EXTL2, SEC61A1, ERAP2, and GALNT14. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting oncogenesis, more preferably by decreasing or inhibiting oncogenesis associated with at least one gene selected from the group consisting of CCND1, CBL, CXCL1, CRKL, MAX, KCNMA1, TBL1XR1, GNAI3, YWHAZ, RAC2, ETS1, PTCH1, MAPK8, LAMC2, PIK3R1, CDK6, CBL, APPL1, GNAI3, PDE3A, TGFB2, AB12, MAX, ITGB3, LOX, CXCL5, ARPCS, PPARGC1A, and THBS4, even more preferably selected from CRKL, TGFB2, YWHAZ, ETS1, MAPK8, and CDK6, most preferably from YWHAZ, ETS1, MAPK8, and CDK6. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by decreasing or inhibiting dysfunctional wound healing, more preferably by decreasing or inhibiting dysfunctional wound healing associated with at least one gene selected from the group consisting of NOTCH2, KCNMA1, CXCL1, ITGB3, PLAU, CCND1, ZMIZ1, ELK3, YWHAZ, IL11, PLAUR, LOX, CTGF, and TGFB2, even more preferably selected from TGFB2, NOTCH2, PLAU, YWHAZ, and PLAUR, most preferably from NOTCH2, PLAU, YWHAZ, and optionally PLAUR. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer by increasing or promoting immune activation, preferably immune activation associated with an immune response against cancer, more preferably by increasing or promoting immune activation associated with at least one gene selected from the group consisting of NOTCH2, LAT2, CRKL, LRRC8A, YWHAZ, PIK3R1, IRF1, TGFB2, IL11, UNG, CDK6, and HPRT1, even more preferably selected from CRKL, TGFB2, NOTCH2, YWHAZ, and CDK6, most preferably from NOTCH2, YWHAZ, and CDK6. Expression or activity of the gene is preferably reduced by the composition for use according to the invention or by the miRNA for use according to the invention.
The invention also provides a T cell obtained from a subject treated with a miRNA for use according to the invention or with a composition for use according to the invention. Such a T cell can be for use in the treatment of cancer as described elsewhere herein. In its use, the T cell is preferably previously obtained from a subject treated with a miRNA for use according to the invention or with a composition for use according to the invention. The T cell is preferably from a human subject. It is preferably for use as a vaccine, or for preventing recurrence or metastasis of cancer.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of a cancer associated with at least one gene selected from the group consisting of CDK6, EIF4B, ETS1, IL17RD, MCL1, MAPK8, NOTCH2, NT5E, PLAU, PLAUR, TNFRSF21, and YWHAZ, more preferably selected from NOTCH2, NT5E, PLAU, PLAUR, and YWHAZ.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of a cancer associated with at least one gene selected from the group consisting of CDK4, CDK6, CRKL, NT5E, HMGB1, IL17RD, KRAS, KIT, HDAC3, RTK2, TGFB2, TNFRSF21, PLAU, NOTCH1, NOTCH2, and YAP1. These genes have known involvement in anti-tumour immunity.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of a cancer associated with at least one gene selected from the group consisting of ETS1, YWHAZ, MPP2, PLAU, CDK4, CDK6, EIF4B, RAD51, CCNA2, STMN1, and DCAF7. These genes are involved in regulation of the cell cycle.
In preferred embodiments, the composition for use according to the invention or the miRNA for use according to the invention is for use in treatment, prevention, delay, or amelioration of cancer, wherein a preferred cancer is a cancer selected from the group consisting of colon cancer such as colon carcinoma, lung cancer such as lung carcinoma, melanoma, lymphoma such as reticulum cell sarcoma, pancreas cancer such as pancreatic adenocarcinoma, liver cancer such as hepatocarcinoma or hepatoma, breast cancer such as breast carcinoma, prostate cancer, kidney cancer such as renal adenocarcinoma, carcinoma such as adenocarcinoma or colon, lung, liver, pancreas, kidney, or breast carcinoma, and adenocarcinoma such as pancreatic or renal adenocarcinoma. A more preferred cancer is a cancer selected from the group consisting of colon cancer such as colon carcinoma, lung cancer such as lung carcinoma, melanoma, lymphoma such as reticulum cell sarcoma, pancreas cancer such as pancreatic adenocarcinoma, liver cancer such as hepatocarcinoma, breast cancer such as breast carcinoma, prostate cancer, carcinoma such as adenocarcinoma or colon, lung, liver, pancreas, or breast carcinoma, and adenocarcinoma such as pancreatic adenocarcinoma. An even more preferred cancer is a cancer selected from the group consisting of colon cancer such as colon carcinoma, lung cancer such as lung carcinoma, melanoma, lymphoma such as reticulum cell sarcoma, and carcinoma such as colon or lung carcinoma.
In further preferred embodiments, the miRNA for use according to the invention is for use in the treatment of cancer wherein the composition is combined with a further chemotherapeutic agent such as sorafenib. This is referred to hereinafter as a combination according to the invention. A combination according to the invention is preferably for use as described above for the composition for use according to the invention.
A combination according to the invention is a combination comprising a composition for use according to the invention or the miRNA for use according to the invention and comprising a chemotherapeutic agent such as a kinase inhibitor drug suitable for the treatment of cancer, for example such as a combination comprising a composition for use according to the invention and comprising sorafenib, or for example comprising a miRNA for use according to the invention and comprising sorafenib.
Suitable chemotherapeutic agents are kinase inhibitor drugs such as sorafenib or B-raf inhibitors or MEK inhibitors or RNR inhibitors or AURKB inhibitors. A preferred B-raf inhibitor is vemurafenib and/or dabrafenib. A preferred MEK inhibitor is trametinib and/or selumetinib. A preferred RNR inhibitor is selected from the group consisting of gemcitabine, hydroxyurea, clolar clofarabine and triapine
B-raf inhibitors are compounds that specifically inhibit the B-raf protein, for which a mutated form of the BRAF gene encodes. Several mutations of the BRAF gene are known to cause melanoma, and specific compounds have been developed which inhibit the mutated form of the B-raf protein. B-raf inhibitors are known in the art and include, but are not limited to vemurafenib, dabrafenib, trametinib, GDC-0879, PLX-4720, sorafenib, SB590885, PLX4720, XL281 and RAF265. B-raf inhibitors are e.g. described in Wong K. K., et al. One B-raf inhibitor may be used or together with other B-raf inhibitors in a combination according to the invention. Preferred B-raf inhibitors to be used in the present invention are vemurafenib, dabrafenib or a mixture of vemurafenib and dabrafenib. Vemurafenib is also known as RG7204 or N-(3-{[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane-1-sulfonamide, and marketed as Zelboraf. Dabrafenib is also known as N-{3-[5-(2-aminopyrimidin-4-yl)-2-(1,1-dimethylethyl)thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide.
MEK inhibitors are compounds that specifically inhibit a MEK protein. Several MEK inhibitors are known in the art and include, but are not limited to trametinib (GSK1120212), selumetinib (AZD-6244), XL518, CI-1040, PD035901. Trametinib is also known as N-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl)phenyl)acetamide. Selumetinib is also known as: 6-(4-bromo-2-chlorophenylamino)-7-fluoro-N-(2-hydroxyethoxy)-3-methyl-3Hbenzo[d]imidazole-5-carboxamide. MEK inhibitors are e.g. described in Wong, K. K. (PMID: 19149686). One MEK inhibitor may be used or together with other MEK inhibitors in a combination according to the invention. Several MEK inhibitors is synonymous with several distinct MEK inhibitors. Prefered MEK inhibitors to be used in the present invention are trametinib and/or selumetinib.
RNR and/or AURKB inhibitors are compounds that specifically inhibit RNR and/or AURKB proteins. RNR is a ribonucleotide reductase (RNR) and as such is the only enzyme responsible for the de novo conversion of ribonucleoside diphosphate (NDP) to deoxyribonucleoside diphosphate (dNDP) (Zhou et al. 2013). RNR is the key regulator of intracellular dNTP supply. Maintenance of a balanced dNTP pool is a fundamental cellular function because the consequences of imbalance in the substrates for DNA synthesis and repair include mutagenesis and cell death. Human RNR is composed of a subunits (RRM1) that contain the catalytic site and two binding sites for enzyme regulators and b subunits (RRM2) with a binuclear iron cofactor that generates the stable tyrosyl radical necessary for catalysis. An inhibitor of RNR may inhibit RRM1 and/or RRM2. Preferred RNR inhibitors are selected from the group consisting of gemcitabine, hydroxyurea, clolar clofarabine and triapine.
AURKB (Aurora B kinase) is a protein that functions in the attachment of the mitotic spindle to the centromere. Chromosomal segregation during mitosis as well as meiosis is regulated by kinases and phosphatases. The Aurora kinases associate with microtubules during chromosome movement and segregation. In cancerous cells, over-expression of these enzymes causes unequal distribution of genetic information, creating aneuploid cells, a hallmark of cancer.
A chemotherapeutic agent is a drug that is able to induce or promote an anti-cancer effect as defined herein. A preferred chemotherapeutic agent is a kinase inhibitor or an RNR inhibitor or an AURKB inhibitor. Examples of such inhibitors are compounds that specifically inhibit the RNR and/or the AURKB proteins. To evaluate the ability of a therapeutic compound to inhibit RNR and/or AURKB proteins, one can perform western blotting with RNR (RRM1 and/or RRM2) or AURKB protein as read-out. Cells are plated in 6-well plates and treated for 72 hours at 0.01, 0.1 and 1 μM of said compound. After treatment cells are scraped into a lysis buffer as a RIPA lysis buffer. Equal amounts of protein extracts are separated by using 10% SDS PAGE, and then transferred to a polyvinylidene difluoride membrane. After blocking for 1 hour in a Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk, the membrane is probed with a RNR (i.e. RRM1 and/or RRM2) and/or a AURKB primary antibody, followed by a secondary antibody conjugated to horseradish peroxidase for chemiluminescent detection on film. Tubulin is used as loading control. A preferred RRM2 antibody used is from Santa Cruz (product #sc-10846) and/or a preferred AURKB antibody is from Cell Signalling (product #3094). The evaluation of the therapeutic ability of said RNR and/or AURKB inhibitor may also be assessed at the RNA level by carrying out a Nothern blot or by PCR.
Preferred combinations according to the invention comprise:

i) a composition for use according to the invention or a miRNA for use according to the invention, and
ii) at least one chemotherapeutic agent selected from the group consisting of
- a. receptor tyrosine kinase inhibitors such as VEGF receptor inhibitors, for example axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, or vandetanib, preferably suntinib, sorafenib, or pazopanib, more preferably sorafenib;
- b. platinum-based cell-cycle nonspecific antineoplastic agents, for example carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, or satraplatin, preferably cisplatin or carboplatin or oxaliplatin;
- c. taxanes, for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel or docetaxel, more preferably paclitaxel or docetaxel;
- d. pyrimidine-based antimetabolites, for example fluorouracil, capecitabine, doxifluridine, tegafur, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, or decitabine, preferably fluorouracil or gemcitabine or capecitabine;
- e. vinca alkaloids, for example vinblastine, vincristine, vinflunine, vindesine, or vinorelbine, preferably vinorelbine or vinblastine;
- f. folic acid antimetabolites, aminopterin, methotrexate, pemetrexed, pralatrexate, or raltitrexed, preferably pemetrexed or methotrexate;
- g. anthracyclins, for example aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin, preferably to doxorubicin;
- h. nonclassical cell-cycle nonspecific antineoplastic agents, for example procarbazine, dacarbazine, temozolomide, altretamine, mitobronitol, or pipobroman, preferably dacarbazine or temozolomide;
- i. taxanes, for example cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, or tesetaxel, preferably paclitaxel such as albumin-bound paclitaxel;
- j. topoisomerase inhibitors, for example camptothecin, cositecan, belotecan, gimatecan, exatecan irinotecan, lurtotecan, silatecan, topotecan, rubitecan, preferably irinotecan;
- k. trifluridine or tipiracil, or a combination of trifluridine and tipiracil;
- l. intercalating crosslinking agents, for example actinomycin, bleomycin, mitomycins, plicamycin, preferably bleomycin or mitomycin;
- m. anthracenediones, for example mitoxantrone or pixantrone, preferably mitoxantrone; and
- n. alkylating antineoplastic agents, for example estrogen-based alkylating antineoplastic agents such as alestramustine, atrimustine, cytestrol acetate, estradiol mustard, estramustine, estromustine, stilbostat; or phenestrol, preferably estramustine.

In preferred embodiments, a composition for use according to the invention or a miRNA for use according to the invention is for use in the treatment of cancer, wherein the composition increases the immune response to cancer cells. This may mean that it initiates an immune response in cases where no immune response was present.
In more preferred embodiments for increasing immune response, the composition for use according to the invention or a miRNA for use according to the invention is for increasing the production of immune system activating cytokines, such as IL-2. Preferably, cytokine production is increased by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more, and is preferably detected through FACS. Immune system activating cytokines are increased in a 4T1 mouse model for triple negative breast cancer (TNBC) after one week of treatment. The increase in cytokines leads to increased immune suppression of cancers, and can lead to immune suppression or partial immune suppression of cancers that would otherwise not be susceptible to immune suppression. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for increasing T cell function, such as increasing production of IFNγ and IL-2.
In more preferred embodiments for increasing immune response, the composition for use according to the invention or a miRNA for use according to the invention is for decreasing regulatory T cell population. Regulatory T cells (Tregs) are immunosuppressive T regulatory cells, and decreasing Tregs increases the immune response to a cancer. Preferably, Tregs are decreased by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Decrease of Tregs can be determined via the determination of FOXP3 or LAG3. This effect is preferably in parallel with increased cytokine production as described above.
Recruitment of CD8+ T effector cells is increased in a 4T1 mouse model for triple negative breast cancer (TNBC) after two weeks of treatment, and T cell function is induced, while Treg population is decreased. Accordingly, in preferred embodiments for increasing immune response, the composition for use according to the invention or a miRNA for use according to the invention is for increasing T cell frequency. Preferably, such an increase is by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Such an increase can be determined by measuring CD8. In preferred embodiments for increasing immune response, the composition for use according to the invention or a miRNA for use according to the invention is for inducing T cell function, preferably for inducing T cell function by inducing IFNγ production. Most preferably, the composition for use according to the invention or a miRNA for use according to the invention is for increasing T cell frequency and simultaneously inducing T cell function, preferably while simultaneously decreasing regulatory T cell population. Tumours with decreased Tregs and with increased CD8+ T effector cells are referred to as ‘hot’ tumours, which are tumours that do not have an immunosuppressed microenvironment. Conversely, tumours in an immunosuppressed microenvironment are referred to as ‘cold’ tumours.
Additionally, compositions according to the invention can reduce expression of immune suppressive target genes such as ENTPD1 (CD39) or TIM-3. Such a reduction is preferably by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. TIM-3 or ENTPD1 expression can be determined via qPCR. ENTPD1 is an ectonucleotidase that catalyses the hydrolysis of γ- and δ-phosphate residues of triphospho- and diphosphonucleosides to the monophosphonucleoside derivative. It has an immune suppressive role through its generation of high amounts of adenosine. Reduction of ENTPD1 expression increases the immune response to tumour cells. TIM-3 is also known as hepatitis A virus cellular receptor 2 (HAVCR2), and is an immune checkpoint, an inhibitory receptor acting as an immune-suppressive marker. TIM-3 is mainly expressed on activated CD8+ T cells and suppresses macrophage activation. Reduction of TIM-3 expression increases the immune response to tumour cells. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for reducing expression of ENTPD1 or of TIM-3 or for reducing expression of ENTPD1 and TIM-3.
The positive effect of compositions according to the invention and miRNA for use according to the invention on the immune system as it relates to tumour cells and cancer cells leads to the invention being suitable for preventing the growth of new tumours, preventing metastasis, or reducing the growth of tumours that have been removed in size, for example through surgery. For example treatment with a composition for use according to the invention reduces the regrowth of surgically excised tumours, and reduces metastasis of such tumours, increasing survival in affected subjects. A tumour from which metastases derive is referred to as a primary tumour. Moreover, subjects with a particular tumour type that had been treated with a composition for use according to the invention or with a miRNA for use according to the invention show limited tumour take when re-challenged with new tumour cells of the same type that had already been treated. After the limited tumour take, the tumour fully regresses. When challenged with a different tumour type, the tumour fully takes, but also subsequently regresses entirely.
Accordingly, in preferred embodiments the compositions according to the invention and miRNA for use according to the invention are for use as a medicament for preventing, reducing, or delaying cancer or metastatic cancer. In this context, preferred cancers are breast cancer, carcinoma, and liver cancer, more preferably breast cancer and liver cancer.
Accordingly, in preferred embodiments the compositions according to the invention and miRNA for use according to the invention are for use as a cancer vaccine, preferably for use as a cancer vaccine for the prevention or treatment of cancer. Such vaccines are preferably for preventing or reducing regrowth or recurrence of primary tumours. Preferably, regrowth is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In another use, such vaccines are preferably for reducing or treating metastatic cancer. Preferably, metastatic cancer is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more, or motility of cancer cells is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In this context, preferred cancers are breast cancer, carcinoma, and liver cancer, more preferably breast cancer and liver cancer.
Accordingly, in preferred embodiments the compositions according to the invention and miRNA for use according to the invention are for use as a medicament, wherein the medicament is for the prevention, reduction, or treatment of metastatic cancer, preferably wherein the primary tumour has been surgically excised or has regressed, more preferably wherein the primary tumour has been surgically excised. Preferably, metastatic cancer is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In this context, preferred cancers are breast cancer, carcinoma, and liver cancer, more preferably breast cancer and liver cancer.
Accordingly, in preferred embodiments the compositions according to the invention and miRNA for use according to the invention are for use as a medicament, wherein the medicament is for the prevention, reduction, or treatment of regrowth or recurrence of a cancer after surgical excision. Preferably, regrowth or recurrence is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In this context, preferred cancers are breast cancer, carcinoma, and liver cancer, more preferably breast cancer and liver cancer.
Accordingly, in preferred embodiments the compositions according to the invention and miRNA for use according to the invention are for use as a medicament, wherein the medicament is for the prevention, reduction, or treatment of regrowth or recurrence of a cancer after said cancer has regressed or has been successfully treated. Preferably, regrowth or recurrence is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In this context, preferred cancers are breast cancer, carcinoma, and liver cancer, more preferably breast cancer and liver cancer.
In preferred embodiments, the composition according to the invention or a miRNA according to the invention is for use in inducing immunogenic cell death. Preferably this use encompasses both the killing of tumor cells and the stimulation of the maturation of dendritic cells. miRNA-193a as defined herein was found to be suitable for inducing immunogenic cell death.
In preferred embodiments, the composition (for use) according to the invention or a miRNA (for use) according to the invention is for promoting the maturation of dendritic cells, preferably for promoting the maturation of dendritic cells from monocytes. The dendritic cells (or their precursor monocytes) will mature, particularly at an enhanced rate, when in the proximity of cells that comprise the miRNA for use according to the invention, for instance because of transfection or because of enhanced expression. Maturation of dendritic cells can be determined by monitoring expression of surface markers, preferably of CD80 or MHC II. Preferably, dendritic cells express at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, of 9.5 times as much surface markers after a subject has been treated with a miRNA for use according to the invention, as compared to a placebo control. Preferred subjects to be treated in this context are subjects who suffer from a condition as described elsewhere herein, and who also suffer from an impaired immune system. miRNA-193a as defined herein was found to be suitable for promoting the maturation of dendritic cells.
In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for inhibiting proliferation of tumour cells. Compositions according to the invention can reduce K-RAS and MCL1 expression, leading to a reduced proliferation of tumour cells. K-RAS, also known as KRAS, K-ras, Ki-ras, is a proto-oncogene known in the art. MCL1 is also known as induced myeloid leukaemia cell differentiation protein Mcl-1. It can enhance cancer cell survival by inhibiting apoptosis. Both K-RAS and MCL1 enhance proliferation of cancer cells. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for reducing expression of K-RAS or of MCL1 or for reducing expression of K-RAS and MCL1. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for reducing expression of K-RAS and MCL1 and ENTPD1 and TIM-3.
Inhibition of proliferation is preferably via induction of apoptosis. Compositions according to the invention induce apoptosis in cancer cells through caspase activation and PARP inactivation through PARP cleavage. Preferred caspase activation is activation of caspase 3/7. PARP is also known as poly (ADP-ribose) polymerase and refers to a family of proteins involved in programmed cell death. It is cleaved in vivo by caspase 3 and by caspase 7, which triggers apoptosis. Cleavage of PARP can be determined through blotting techniques, and caspase activation can be assayed by determining PARP cleavage through blotting, or by qPCR. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for inducing apoptosis in cancer cells. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for activating caspase 3 and caspase 7. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for inactivating PARP. Preferably, PARP is inactivated by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Inactivation of PARP can be monitored by blotting techniques, detecting the smaller fragments of the uncleaved enzyme. Preferably, caspase activity is increased by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more.
In further preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for reducing expression of at least one of the genes selected from the group consisting of K-RAS, MCL1, ENTPD1, TIM-3, c-Kit, CyclinD1, and CD73. c-Kit is a proto-oncogene also known as tyrosine-protein kinase Kit or CD117, and codes for a receptor tyrosine kinase protein. Cyclin D1 overexpression correlates with early cancer onset and tumour progression. CD73 is also known as 5′-nucleotidase (5′-NT), and as ecto-5′-nucleotidase. The enzyme encoded by CD73 is ecto-5-prime-nucleotidase (5-prime-ribonucleotide phosphohydrolase; EC 3.1.3.5) and catalyzes the conversion at neutral pH of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. Expression of such genes is preferably reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more, which can for example be determined via qPCR techniques.
In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for regulating the adenosine A2A receptor pathway. The adenosine A2A receptor, also known as ADORA2A, is an adenosine receptor that can suppress immune cells. The activity of compositions according to the invention in reducing expression of CD73 and/or of ENTPD1, as described above, interferes with the A2A receptor pathway, reducing immune suppression. This leads to an anti-tumour effect because tumour cells ability to escape immune surveillance is reduced. In preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for increasing the susceptibility of tumour cells to immune surveillance. Such an increase preferably leads to a reduction of tumour volume of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. In more preferred embodiments, the composition for use according to the invention or a miRNA for use according to the invention is for increasing the susceptibility of tumour cells to immune surveillance, while increasing recruitment of CD8+ T effector cells, preferably while decreasing Tregs, such as through reducing expression of LAG3 or of FoxP3, or of both. Increased susceptibility to immune surveillance preferably leads to reduced tumour volume.
The inventors have found that miRNA-193a modulates several pathways and genes. This activity of miRNA-193a can be used for treating conditions associated with those pathways or genes. Accordingly, in preferred embodiments is provided the miRNA-193a or a source thereof for use according to the invention, wherein the miRNA-193a modulates expression of a gene selected from the group consisting of RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1, and optionally HMGB2, preferably from the group consisting of RPS6KB2, KRAS, PDGFRB, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, MCL1, more preferably selected from PDPK1 or INPPL1. This modulation is preferably downregulation. In preferred embodiments PDPK1 is modulated, preferably downregulated by the miRNA. In preferred embodiments INPPL1 is modulated, preferably downregulated by the miRNA. In one embodiment the gene is HMGB1, which is preferably downregulated.
Modulation is defined elsewhere herein. Upregulation refers to an increased expression, which can refer to an increased transcription, production of mRNA, translation, production of gene product, and/or activity of gene product. Downregulation refers to a decreased expression, can refer to a decreased transcription, production of mRNA, translation, production of gene product, and/or activity of gene product. Preferably, upregulation and downregulation refer to transcription of production of mRNA. In other preferred embodiments upregulation and downregulation refer to activity of gene product. Upregulation and downregulation are preferably as compared to a reference, such as to a healthy cell, or such as to an untreated cell, when cultivated under otherwise identical conditions. For example, when miRNA-193a is used for downregulation of INPPL1 in a cell, then miRNA-193a preferably decreases INPPL1 expression in that cell as compared to a cell (of the same type) that has not been contacted with miRNA-193a. The change in expression is preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 125, 150, 200, 250% or more, more preferably at least 50% or more, even more preferably at least 100% or more. In case of downregulation, optionally there is no longer any detectable expression after downregulation.
Compositions for use according to the invention and miRNA for use according to the invention promote cell cycle arrest in tumour cells. In preferred embodiments, the miRNA for use according to the invention or the composition for use according to the invention are for use in the treatment of cancer, wherein the use is for inducing cell cycle arrest. Cell cycle arrest profiles can be measured for example by performing either nuclei imaging or flow cytometry, preferably as demonstrated in the examples. In this context, cell cycle arrest is preferably the induction of a G2/M or a SubG1 cell cycle arrest profile. Preferably, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more tumour cells undergo cell cycle arrest. Preferably, when the miRNA for use according to the invention is for treating PTEN-deficient melanoma, liver cancer, carcinoma, lung cancer, or pancreas cancer, the miRNA for use according to the invention is for increasing cell cycle arrest profiles.

Composition

The invention also relates to compositions comprising the miRNA for use according to the invention, wherein the composition is for that same use. Such a composition comprises a miRNA-193a or a source thereof as for use according to the invention. It is referred to hereinafter as a composition for use according to the invention. Preferably such compositions are pharmaceutical compositions. Such compositions further preferably comprise a pharmaceutically acceptable solvent, or a pharmaceutically acceptable excipient, or a pharmaceutically acceptable diluent, or a pharmaceutically acceptable carrier.
Preferred compositions for use according to the invention comprise a miRNA-193a or a source thereof, preferably wherein the miRNA-193a is a miRNA193a molecule, an isomiR, or a mimic thereof. More preferably, compositions for use according to the invention comprise a miRNA-193a or a source thereof, wherein the miRNA-193a is a miRNA-193a molecule, an isomiR, or a mimic thereof, and is an oligonucleotide with a seed sequence comprising at least 6 of the 7 nucleotides of the seed sequence represented by SEQ ID NO: 22. Highly preferred compositions comprise nanoparticles as later defined herein.
In preferred embodiments, this aspect provides the composition for use according to the invention, further comprising a further miRNA or precursor thereof, wherein the further miRNA is selected from the group consisting of miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or an isomiR thereof, or a mimic thereof.
The inventors have surprisingly found that a nanoparticle formulation comprising a diamino lipid provides excellent results when used as compositions for use according to the invention. Accordingly, in preferred embodiments the composition for use according to the invention is a nanoparticle composition, the nanoparticle comprising a diamino lipid and a miRNA-193a or a source thereof as defined above, wherein the diamino lipid is of general formula (I)
wherein

Such a composition is referred to hereinafter as a nanoparticle composition for use according to the invention. In the context of this application, a nanoparticle is a particle with dimensions in the nanometer range, or in some cases in the micrometer range. Preferably, a nanoparticle is as least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nanometer in diameter, where a diameter is preferably an average diameter of a population of nanoparticles. Preferably, a nanoparticle is at most 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 5000, or 10000 nanometer in diameter. More preferably, nanoparticles have an average diameter of 40-300 nm, even more preferably of 50-200 nm, even more preferably of 50-150 nm, most preferably of 65-85 nm, such as about 70 nm.
Nanoparticle compositions for use according to the invention comprise lipid nanoparticles that further comprise an oligonucleotide. The oligonucleotide can be seen as the cargo or the payload of the nanoparticle. Accordingly, the nanoparticles can for example be micelles, liposomes, lipoplexes, unilamellar vesicles, multilamellar vesicles, or cross-linked variants thereof. It is preferred that the nanoparticles are micelles, liposomes, or lipoplexes. When reference is made to the composition of the nanoparticles, reference to the diamino lipid and optional further excipients is intended, and no reference to any cargo substances is intended. As a non-limiting example, when the nanoparticle is said to comprise 50 mol % of the diamino lipid and 50 mol % of other excipients, the molar percentages only relate to the diamino lipid and those other excipients; the oligonucleotide molar fraction or the molar fraction of solvents is not taken into account.
When the invention relates to a composition comprising more than one miRNA molecule, isomiR, mimic, or source thereof it is encompassed that each miRNA molecule, isomiR, mimic, or source thereof may be present each in a separate composition. Each composition can be sequentially or simultaneously administered to a subject, or mixed prior to use into a single composition. Alternatively, it is also encompassed that more than one miRNA molecules, isomiRs, mimics, or sources thereof is present in a single composition as defined herein.
The nanoparticle compositions for use according to the invention comprises a diamino lipid of general formula (I), but it may also comprise further lipids. In preferred embodiments, the diamino lipid is the most prevalent lipid in the nanoparticle by molar percent. As used herein, the term lipid refers to substances that are soluble in nonpolar solvents such as CH₂Cl₂. The diamino lipids used in the invention have three tails linked to a spacer and thus resemble naturally occurring triglyceride lipids. Several such lipids are known (U.S. Pat. No. 8,691,750).
The diamino lipid of general formula (I) comprises two tertiary amines that are separated by an aliphatic spacer of varying length. The spacer helps determine the headgroup size of the lipid. n can be 0, 1, or 2, so the spacer is in effect an 1,2-ethylene, n-1,3-propylene, or n-1,4-butylene spacer. In particular preferred embodiments, n is 0. In particular preferred embodiments, n is 1. In particular preferred embodiments, n is 2. It is most preferred that n is 1. Accordingly, in preferred embodiments the invention provides a nanoparticle composition for use according to the invention, wherein the diamino lipid is of general formula (I) wherein n is 1. Accordingly, in preferred embodiments the invention provides a nanoparticle composition for use according to the invention, wherein the diamino lipid is of general formula (I-1)

- Wherein T¹, T², and T³are each independently a C₁₀-C₁₈chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.

T¹, T², and T³can be seen as the tails of the lipid, and are aliphatic C₁₀-C₁₈with optional unsaturations and up to four optional substitutions. T¹, T², and T³can be independently selected, or the same choice can be made for two or three of T¹, T², and T³. In preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, wherein the diamino lipid is of general formula (I) wherein T¹, T², and T³are identical. Identical should not be so narrowly construed as to imply that the natural abundance of isotopes should be contemplated—identical should preferably only refer to the molecular structure as would be represented in a drawn structural formula.
Longer chains will generally lead to more rigid lipid membranes. In this application the number in C₁₀-C₁₈refers to the longest continuous chain that can be determined, and not to the total C content. As a non-limiting example, an n-dodecyl chain with an n-propyl substitution at a δ-position comprises 15 C atoms but is a C₁₂chain because the longest continuous chain has a length of 12 C atoms. Unsaturations can lead to less rigid membranes if the unsaturation is cis in the chain, bending it. A preferred unsaturation is cis. In preferred embodiments, T¹, T², and T³contain zero, one, two, three, or four unsaturations. In more preferred embodiments, T¹, T², and T³contain one, two, three, or four unsaturations. In even more preferred embodiments, T¹, T², and T³contain one, two, or three unsaturations, preferably three unsaturations.
The optional substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy A preferred optional substitution is a C₁-C₄alkyl, more preferably a C₁-C₂alkyl, most preferably methyl (—CH₃). There are zero, one, two, three, or four of such substitutions, which means that substitutions can be absent. As such the substitutions are optional. Preferably, there are zero, one, two, or three such substitutions.
In preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₆chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy. In more preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₄chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy. Most preferably, T¹, T², and T³are each independently a C₁₂chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.
In preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₈chain with one, two, three, or four unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.
In preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₈chain with one, two, or three unsaturations and with one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.
In preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₈chain with one, two, or three unsaturations and with one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl.
In preferred embodiments, T¹, T², and T³are each independently a C₁₀-C₁₄chain with one, two, or three unsaturations and with one, two, or three substitutions, wherein the substitutions are selected from the group consisting of C₁-C₂alkyl.
Preferred embodiments for T¹, T², and T³are (with a name in systematic C_nnumbering, wherein a number after a colon (as in C1_2:3) indicates the degree of unsaturation) (2E,6E)-farnesyl (C_12:3), lauryl (C₁₂), tridecyl (C₁₃), myristryl (Cu), pentadecyl (C₁₅), cetyl (C₁₆), margaryl (C₁₇), stearyl (C₁₈), α-linolenyl (C_18:3), γ-linolenyl (C_18:3), linoleyl (C_18:2), stearidyl (C_18:4), vaccenyl (C_18:1), oleyl (C_18:1), elaidyl (C_18:1), palmitoleyl (C_16:1), (2E,6Z)-farnesyl, (2Z,6E)-farnesyl, (2Z,6Z)-farnesyl, and 3,7,11-trimethyldodecyl.
Accordingly, in preferred embodiments this aspect provides the nanoparticle composition for use according to the invention, wherein the diamino lipid is of general formula (I) wherein T¹, T², and T³are each independently selected from the group consisting of farnesyl, lauryl, tridecyl, myristryl, pentadecyl, cetyl, margaryl, stearyl, α-linolenyl, γ-linolenyl, linoleyl, stearidyl, vaccenyl, oleyl, elaidyl, palmitoleyl, and 3,7,11-trimethyldodecyl. Preferably, T¹, T², and T³are each independently selected from the group consisting of farnesyl, lauryl, tridecyl, myristryl, pentadecyl, cetyl, α-linolenyl, γ-linolenyl, linoleyl, stearidyl, oleyl, palmitoleyl, and 3,7,11-trimethyldodecyl. More preferably, T¹, T², and T³are each independently selected from the group consisting of farnesyl, lauryl, tridecyl, myristryl, stearidyl, palmitoleyl, and 3,7,11-trimethyldodecyl. Even more preferably, T¹, T², and T³are each independently selected from the group consisting of farnesyl, lauryl, tridecyl, myristryl, and 3,7,11-trimethyldodecyl. Even more preferably, T¹, T², and T³are each independently selected from the group consisting of farnesyl, lauryl, and 3,7,11-trimethyldodecyl. Most preferably, T¹, T², and T³are each independently farnesyl, such as (2E,6E) farnesyl, (2E,6Z) farnesyl, (2Z,6E) farnesyl, or (2Z,6Z) farnesyl; preferably they are each (2E,6E) farnesyl.
Farnesyl is also known as 3,7,11-trimethyldodeca-2,6,10-trienyl and is an unsaturated linear C₁₂chain; it can be (2E,6E), (2E,6Z), (2Z,6E), or (2Z,6Z); preferably it is (2E,6E). Lauryl is also known as dodecyl and is a saturated linear C₁₂chain. Tridecyl is a saturated linear C₁₃chain. Myristryl is also known as tetradecyl and is a saturated linear C₁₄chain. Pentadecyl is a saturated linear C₁₅chain. Cetyl is also known as palmityl and is a saturated linear C₁₆chain. Margaryl is also known as heptadecyl and is a saturated linear C₁₇chain. Stearyl is also known as octadecyl and is a saturated linear C₁₈chain. α-linolenyl is also known as (9Z,12Z,15Z)-9,12,15-octadecatrienyl and is an unsaturated linear C₁₈chain. γ-linolenyl is also known as (6Z,9Z, 12Z)-6,9,12-octadecatrienyl and is an unsaturated linear C₁₈chain. Linoleyl is also known as (9Z,12Z)-9,12-octadecadienyl and is an unsaturated linear C₁₈chain. Stearidyl is also known as (6Z,9Z,12Z,15Z)-6,9,12,15-octadecatetraenyl and is an unsaturated linear C₁₈chain. Vaccenyl is also known as (E)-octadec-11-enyl and is an unsaturated linear C₁₈chain. Oleyl is also known as (9Z)-octadec-9-enyl and is an unsaturated linear C₁₈chain. Elaidyl is also known as (9E)-octadec-9-enyl and is an unsaturated linear C₁₈chain. Palmitoleyl is also known as (9Z)-hexadec-9-enyl and is an unsaturated linear C₁₆chain. 3,7,11-trimethyldodecyl is saturated farnesyl and is a saturated linear C₁₂chain.
The composition can further comprise solvents and/or excipients, preferably pharmaceutically acceptable excipients. Preferred solvents are aqueous solutions such as pharmaceutically acceptable buffers, for example PBS or citrate buffer. A preferred citrate buffer comprises 50 mM citrate at pH 2.5-3.5 such as pH 3, preferably set using NaOH. A preferred PBS is at pH 7-8 such as pH 7.4. PBS preferably does not comprise bivalent cations such as Ca²⁺ and Mg²⁺. Another preferred pharmaceutically acceptable excipient is ethanol. Most preferably, the composition comprises a physiological buffer such as PBS or a Good's buffer or Hepes-buffered saline or Hank's balanced salt solution or Ringer's balanced salt solution or a Tris buffer. Preferred compositions are pharmaceutical compositions. The composition can comprise further excipients. These further excipients can be comprised in the nanoparticles.
In preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, further comprising a sterol, preferably selected from the group consisting of adosterol, brassicasterol, campesterol, cholecalciferol, cholestenedione, cholestenol, cholesterol, delta-7-stigmasterol, delta-7-avenasterol, dihydrotachysterol, dimethylcolesterol, ergocalciferol, ergosterol, ergostenol, ergostatrienol, ergostadienol, ethylcholestenol, fusidic acid, lanosterol, norcholestadienol, β-sitosterol, spinasterol, stigmastanol, stigmastenol, stigmastadienol, stigmastadienone, stigmasterol, and stigmastenone, more preferably cholesterol. More particularly, in preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, wherein the nanoparticles further comprise a sterol, preferably selected from the group consisting of adosterol, brassicasterol, campesterol, cholecalciferol, cholestenedione, cholestenol, cholesterol, delta-7-stigmasterol, delta-7-avenasterol, dihydrotachysterol, dimethylcolesterol, ergocalciferol, ergosterol, ergostenol, ergostatrienol, ergostadienol, ethylcholestenol, fusidic acid, lanosterol, norcholestadienol, β-sitosterol, spinasterol, stigmastanol, stigmastenol, stigmastadienol, stigmastadienone, stigmasterol, and stigmastenone, more preferably cholesterol.
Preferably, such a further comprised sterol is not conjugated to any moiety. Conjugated sterols can also be comprised, as will be explained later herein. As such, both conjugated and unconjugated sterols can be comprised. Unless explicitly indicated otherwise, reference to a sterol is intended as reference to an unconjugated sterol.
When a sterol is comprised in the composition, it is preferably comprised in the nanoparticle, and preferably at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mol % of sterol is comprised; preferably at most 80, 75, 70, 65, 60, 65, 50, 45, 40, 35, or 30 mol % of sterol is comprised. As explained above, this molar percentage only pertains to the substances making up the lipid nanoparticle, and not to solvents or cargo such as oligonucleotides. When a sterol is comprised in the composition, preferably 5 to 70 mol %, 15 to 60 mol %, 25 to 60 mol %, 35 to 60 mol %, 40 to 60 mol %, or 45 to 55 mol % is comprised; more preferably 40 to 60 mol % or 45 to 55 mol % is comprised, most preferably 45 to 55 mol % is comprised, such as 48 mol % or 54 mol %.
In preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, further comprising a phospholipid, preferably selected from the group consisting of distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dilauroyl phosphatidylcholine (DLPC), dioleyl phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), egg phosphatidylcholine (EggPC), soy phosphatidylcholine (SoyPC), more preferably distearoyl phosphatidylcholine (DSPC). More particularly, in preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, wherein the nanoparticles further comprise a phospholipid, preferably selected from the group consisting of distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dilauroyl phosphatidylcholine (DLPC), dioleyl phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), egg phosphatidylcholine (EggPC), soy phosphatidylcholine (SoyPC), more preferably distearoyl phosphatidylcholine (DSPC).
Preferably, such a further comprised phospholipid is not conjugated to any moiety. Conjugated phospholipids can also be comprised, as will be explained later herein. As such, both conjugated and unconjugated phospholipids can be comprised.
When a phospholipid is comprised in the composition, it is preferably comprised in the nanoparticle, and preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol % of phospholipid is comprised; preferably at most 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 mol % of phospholipid is comprised. As explained above, this molar percentage only pertains to the substances making up the lipid nanoparticle, and not to solvents or cargo such as oligonucleotides. When a phospholipid is comprised in the composition, preferably 0 to 40 mol %, 0 to 35 mol %, 0 to 30 mol %, 5 to 30 mol %, 5 to 25 mol %, or 5 to 20 mol % is comprised; more preferably 5 to 20 mol % or 5 to 15 mol % is comprised, most preferably 5 to 15 mol % is comprised, such as 10 mol % or 11 mol %.
In preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, further comprising a conjugate of a water soluble polymer and a lipophilic anchor, wherein:

- i) the water soluble polymer is selected from the group consisting of poly(ethylene glycol) (PEG), poly(hydroxyethyl-l-asparagine) (PHEA), poly-(hydroxyethyl-L-glutamine) (PHEG), poly(glutamic acid) (PGA), polyglycerol (PG), poly(acrylamide) (PAAm), poly(vinylpyrrolidone) (PVP), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), and poly(2-oxazoline) (POx) such as poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx), or copolymers thereof and wherein
- ii) the lipophilic anchor is selected from the group consisting of a sterol, a lipid, and a vitamin E derivative. Preferably, the lipophilic anchor is a lipid, more preferably a diglyceride.

More particularly, in preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, wherein the nanoparticles further comprise a conjugate of a water soluble polymer and a lipophilic anchor as described above. The water soluble polymer generally increases the colloidal stability of the nanoparticles, to which is it linked via the lipophilic anchor. In general, the lipophilic anchor embeds in the lipid bilayer or in the micelle, and thus links the water soluble polymer to the surface of the nanoparticle. The use of such water soluble polymers for this purpose is known in the art (Knop et al., 2010, doi: 10.1002/anie.200902672). A preferred water soluble polymer is poly(ethylene glycol). Preferably, the water soluble polymer has a molecular weight ranging from about 750 Da to about 15000 Da, more preferably from about 1000 Da to about 6000 Da, even more preferably from about 1000 Da to about 3000 Da, most preferably from about 1500 Da to about 3000 Da, such as about 2000 Da. Accordingly, PEG-2000 is a preferred water soluble polymer for use in a conjugate as described above. The water soluble polymer is preferably a linear polymer, and is preferably conjugated at one of its two termini. The other terminus is preferably uncharged at physiological conditions, such as a hydroxyl group or a methyl or ethyl ether. Preferably, the non-conjugated terminus is a methyl ether or a hydroxyl group, most preferably a methyl ether.
The lipophilic anchor to which the water soluble polymer is conjugated generally serves to ensure a connection between the water soluble polymer and the nanoparticle. The particular conjugation between the polymer and the anchor is not important, a skilled person can select any suitable chemical bond such as an ester bond, an amide bond, an ether linkage, a triazole, or any other moiety resulting from conjugating a water soluble polymer to a lipophilic anchor. The use of small linkers is also envisaged, such as succinic acid or glutaric acid. The lipophilic anchor is selected from the group consisting of a sterol, a lipid, and a vitamin E derivative. Preferred sterols are described above. Preferred vitamin E derivatives are tocopherols and tocotrienols such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and corresponding tocotrienols. Preferably, the lipophilic anchor is a lipid, more preferably a diglyceride or a phospholipid. Examples of preferred lipids are described above, examples of preferred diglycerides are distearoylglycerol, preferably 1,2-distearoyl-sn-glycerol, dipalmitoylglycerol, preferably 1,2-dipalmitoyl-sn-glycerol, dioleoylglycerol, preferably 1,2-dioleoyl-sn-glycerol, and diarachidoylglycerol, preferably 1,2-diarachidoyl-sn-glycerol. A most preferred diglyceride is distearoylglycerol, preferably 1,2-distearoyl-sn-glycerol.
Suitable examples of conjugates as described above are (1,2-distearoyl-sn-glycerol)-[methoxy(polyethylene glycol-2000)] ether, (1,2-distearoyl-sn-glycerol)-[methoxy(polyethylene glycol-1500)] ether, (1,2-distearoyl-sn-glycerol)-[methoxy(polyethylene glycol-3000)]ether, (1,2-distearoyl-sn-glycerol)-[hydroxy(polyethylene glycol-2000)]ether, (1,2-distearoyl-sn-glycerol)-[hydroxy(polyethylene glycol-1500)]ether, (1,2-distearoyl-sn-glycerol)-[hydroxy(polyethylene glycol-3000)]ether, (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-2000)carboxylate], (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-1500) carboxylate], (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-3000) carboxylate], (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-2000) carboxylate], (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-1500) carboxylate], (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-3000) carboxylate], (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-2000) carbamate], (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-1500) carbamate], (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-3000) carbamate], (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-2000) carbamate], (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-1500) carbamate], and (1,2-distearoyl-sn-glyceryl)-[hydroxy(polyethylene glycol-3000) carbamate], wherein the stearoyl moieties can optionally be replaced by other fatty acids, preferably by other C₁₀-C₂₀fatty acids. For carbamates and esters as described above, the parent amines and parent alcohols and parent carboxylic acids can also be switched around, for example a PEG-alcohol can be reacted with a carboxylic acid analogue of a diglyceride. Most preferred examples of conjugates are (1,2-distearoyl-sn-glycerol)[methoxy(polyethylene glycol-2000)] ether, which is also known as DSG-PEG (CAS #: 308805-39-2), and its ester analogue (1,2-distearoyl-sn-glyceryl)[methoxy(polyethylene glycol-2000)carboxylate], and its carbamate analogue (1,2-distearoyl-sn-glyceryl)-[methoxy(polyethylene glycol-2000) carbamate] or 1,2-distearoyloxy propylamine 3-N-methoxy(polyethylene glycol)-2000 carbamoyl which is also known as DSA-PEG, and its amide analogue.
When a conjugate as described above is comprised in the composition, it is preferably comprised in the nanoparticle, and preferably at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mol % of conjugate is comprised; preferably at most 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mol % of conjugate is comprised. As explained above, this molar percentage t only pertains to the substances making up the lipid nanoparticle, and not to solvents or cargo such as oligonucleotides. When a conjugate is comprised in the composition, preferably 0 to 4 mol %, 0 to 3 mol %, 0.3 to 3 mol %, 0.5 to 3 mol %, 0.5 to 2.5 mol %, or 1 to 2.5 mol % is comprised; more preferably 0.5 to 2.5 mol % or 0.7 to 2.5 mol % is comprised, most preferably 0.8 to 2.4 mol % is comprised, such as 1 mol % or 2 mol %.
Preferred nanoparticles comprise a diamino lipid and a sterol. Further preferred nanoparticles comprise a diamino lipid and a phospholipid. Further preferred nanoparticles comprise a diamino lipid and a conjugate of a water soluble polymer and a lipophilic anchor. Preferred nanoparticles comprise a diamino lipid and a sterol and a phospholipid. Preferred nanoparticles comprise a diamino lipid and a sterol and a conjugate of a water soluble polymer and a lipophilic anchor. Preferred nanoparticles comprise a diamino lipid and a phospholipid and a conjugate of a water soluble polymer and a lipophilic anchor. Most preferred nanoparticles comprise a diamino lipid and a sterol and a phospholipid and a conjugate of a water soluble polymer and a lipophilic anchor.
In preferred embodiments, this aspect provides the nanoparticle composition for use according to the invention, wherein the nanoparticles comprise:

- i) 20-60 mol % of diamino lipid, and
- ii) 0-40 mol % of phospholipid, and
- iii) 30-70 mol % of a sterol, preferably cholesterol, and
- iv) 0-10 mol % of a conjugate of a water soluble polymer and a lipophilic anchor as defined above.
  In further preferred embodiments the nanoparticles comprise
- i) 25-55 mol % of diamino lipid, and
- ii) 1-30 mol % of phospholipid, and
- iii) 35-65 mol % of a sterol, preferably cholesterol, and
- iv) 0.1-4 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.
  In further preferred embodiments the nanoparticles comprise
- i) 30-50 mol % of diamino lipid, and
- ii) 5-15 mol % of phospholipid, and
- iii) 40-60 mol % of a sterol, preferably cholesterol, and
- iv) 0.5-2.5 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.
  In further preferred embodiments the nanoparticles comprise
- i) about 38-42 mol % of diamino lipid, and
- ii) about 8-12 mol % of phospholipid, and
- iii) about 46-50 mol % of a sterol, preferably cholesterol, and
- iv) about 1.8-2.2 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.

The composition for use according to the invention can advantageously comprise additional therapeutically active agents. In preferred embodiments is provided the composition for use according to the invention, further comprising an additional pharmaceutically active compound, preferably selected from the group consisting of a PP2A methylating agent, an inhibitor of hepatocyte growth factor (HGF), an antibody, a PI3K inhibitor, an Akt inhibitor, an mTOR inhibitor, a binder of a T cell co-stimulatory molecule such as a binder of OX40, and a chemotherapeutic agent. Chemotherapeutic agents are defined later herein.
A PP2A methylating agent can activate PP2A, which in turn activates tumour suppressors such as p53 (see US2007280918). A particularly preferred PP2A methylating agent is betaine (betaine hydrate or also trimethylammonio-2 acetate) or one of its pharmaceutically acceptable salts, in particular betaine citrate. An HGF inhibitor can inhibit HGF, which is coexpressed, often over-expressed, on various human solid tumors including tumors derived from lung, colon, rectum, stomach, kidney, ovary, skin, multiple myeloma and thyroid tissue (see WO2009126842). Preferred HGF inhibitors are truncated HGF proteins such as NKI (N terminal domain plus kringle domain 1; Lokker et al., J. Biol. Chem. 268:17145, 1993); NK2 (N terminal domain plus kringle domains 1 and 2; Chan et al, Science 254:1382, 1991); and NK4 (N-terminal domain plus four kringle domains), which was shown to partially inhibit the primary growth and metastasis of murine lung tumor LLC in a nude mouse model (Kuba et al, Cancer Res. 60:6737, 2000), anti-HGF mAbs such as L2G7 (Kim et al, Clin Cancer Res 12:1292, 2006 and U.S. Pat. No. 7,220,410), HuL2G7 (WO 07115049 A2), the human mAbs described in WO 2005/017107 A2, and the HGF binding proteins described in WO 07143090 A2 or WO 07143098 A2. PI3K inhibitors are widely known. Preferred PI3K inhibitors are GSK2636771 B, GSK2636771, idelalisib, copanlisib, duvelisib, and alpelisib. Akt inhibitors are widely known. Preferred Akt inhibitors are VQD-002, perifosine, miltefosine, MK-2206, AZD5363, and ipatasertib. mTOR inhibitors are widely known. Preferred mTOR inhibitors are sirolimus, everolimus, ridaforolimus, temsirolimus, umirolimus, and zotarolimus. Binder of a T cell co-stimulatory molecule are described in WO2019106605. A preferred such binder is a binder of OX40 such as an antibody against OX40.

Method for Agonizing PTEN

The invention also provided a method for agonising PTEN, the method comprising the step of contacting a cell with a miRNA-193a as defined for use above, or with a composition as defined for use above. Accordingly, the cell is contacted with a miRNA-193a molecule, isomiR, mimic, or source thereof. The method van be an in vivo, in vitro, or ex vivo method, and preferably it is an in vitro or ex vivo method. Agonising PTEN is as defined elsewhere herein, and is preferably increasing expression of PTEN or increasing PTEN protein activity or increasing PTEN protein levels, more preferably it is increasing PTEN protein activity. PTEN activity of levels are preferably increased by at least 5%, more preferably by at least 25%. Ways for contacting a cell are widely known in the art; preferably the miRNA is added to the cell culture medium without further excipients, or it is transfected such as by using transfection reagents.

General Definitions

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value more or less 1% of the value. When moieties or substructures of molecules are said to be identical, the natural abundance distribution of isotopes is not accounted for. The identical nature refers to a structural formula as it would be drawn.
As used herein, mol % refers to molar percentage, which is also known as a mole fraction or a molar fraction or a mole percent or an amount fraction. It relates to the amount in moles of a constituent, divided by the total amount of all constituents in a mixture, also expressed in moles.
In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.
The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use are suitable for use in methods of treatment, for example in a method for treating a condition associated with PTEN-deficiency, preferably a PTEN-deficient cancer, the method comprising the step of administering to a subject a miRNA-193a for use according to the invention, or a composition for use according to the invention.
The present invention has been described above with reference to a number of exemplary embodiments. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims. All citations of literature and patent documents are hereby incorporated by reference.
“Modulate” as used herein, for example with regard to expression of a gene, means to change any natural or existing level of function, for example it means affecting expression by increasing or reducing it. Modulation includes upregulating or agonizing, e.g., signaling, as well as downregulating, antagonizing, or blocking signaling or interactions with a ligand or compound or molecule that happen in the unchanged or unmodulated state. Thus, modulators may be agonists or antagonists. Agonist or antagonist activity can be measured in vitro by various assays know in the art such as, but not limited to, measurement of cell signalling, cell proliferation, immune cell activation markers, and cytokine production, optionally including comparison to unmodulated reference samples. Agonist or antagonist activity can also be measured in vivo by various assays that measure surrogate end points such as, but not limited to the measurement of T cell proliferation or cytokine production.
General Technologies Referred to Herein
MicroRNA molecules (“miRNAs”) are generally 21 to 22 nucleotides in length, though lengths of 17 and up to 25 nucleotides have been reported. Any length of 17, 18, 19, 20, 21, 22, 23, 24, 25 is therefore encompassed within the present invention. The miRNAs are each processed from a longer precursor RNA molecule (“precursor miRNA”). Precursor miRNAs are transcribed from non-protein-encoding genes. A precursor may have a length of at least 50, 70, 75, 80, 85, 100, 150, 200 nucleotides or more. The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved by enzymes called Dicer and Drosha in animals. Dicer and Drosha are ribonuclease III-like nucleases. The processed miRNA is typically a portion of the stem.
The processed miRNA (also referred to as “mature miRNA”) becomes part of a large complex, known as the RNA-Induced Silencing Complex (RISC) complex, to (down or up)-regulate a particular target gene. Examples of animal miRNAs include those that perfectly or imperfectly basepair with the mRNA target, resulting in either mRNA degradation or inhibition of translation respectively (Olsen et al, 1999; Seggerson et al, 2002). SiRNA molecules also are processed by Dicer, but from a long, double-stranded RNA molecule. SiRNAs are not naturally found in animal cells, but they can function in such cells in a RNA-induced silencing complex (RISC) to direct the sequence-specific cleavage of an mRNA target (Denli et al, 2003).
The study of endogenous miRNA molecules is described in U.S. Patent Application 60/575,743. A miRNA is apparently active in the cell when the mature, single-stranded RNA is bound by a protein complex that regulates the translation of mRNAs that hybridize to the miRNA. Introducing exogenous RNA molecules that affect cells in the same way as endogenously expressed miRNAs requires that a single-stranded RNA molecule of the same sequence as the endogenous mature miRNA be taken up by the protein complex that facilitates translational control. A variety of RNA molecule designs have been evaluated. Three general designs that maximize uptake of the desired single-stranded miRNA by the miRNA pathway have been identified. An RNA molecule with a miRNA sequence having at least one of the three designs may be referred to as a synthetic miRNA.
miRNA molecules of the invention can replace or supplement the gene silencing activity of an endogenous miRNA. An example of such molecules, preferred characteristics and modifications of such molecules and compositions comprising such molecules is described in WO2009/091982.
miRNA molecules of the invention or isomiRs or mimics or sources thereof comprise, in some embodiments, two RNA molecules wherein one RNA is identical to a naturally occurring, mature miRNA. The RNA molecule that is identical to a mature miRNA is referred to as the active strand or the antisense strand. The second RNA molecule, referred to as the complementary strand or the sense strand, is at least partially complementary to the active strand. The active and complementary strands are hybridized to create a double-stranded RNA, that is similar to the naturally occurring miRNA precursor that is bound by the protein complex immediately prior to miRNA activation in the cell. Maximizing activity of said miRNA requires maximizing uptake of the active strand and minimizing uptake of the complementary strand by the miRNA protein complex that regulates gene expression at the level of translation. The molecular designs that provide optimal miRNA activity involve modifications of the complementary strand. Two designs incorporate chemical modifications of the complementary strand. The first modification involves creating a complementary RNA with a group other than a phosphate or hydroxyl at its 5′ terminus. The presence of the 5′ modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of the active strand by the miRNA protein complex. The 5′ modification can be any of a variety of molecules including NH₂, NHCOCH₃, biotin, and others. The second chemical modification strategy that significantly reduces uptake of the complementary strand by the miRNA pathway is incorporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that the sugar modifications consistent with the second design strategy can be coupled with 5′ terminal modifications consistent with the first design strategy to further enhance miRNA activities. The third miRNA design involves incorporating nucleotides in the 3′ end of the complementary strand that are not complementary to the active strand. Hybrids of the resulting active and complementary RNAs are very stable at the 3′ end of the active strand but relatively unstable at the 5′ end of the active strand. Studies with siRNAs indicate that 5′ hybrid stability is a key indicator of RNA uptake by the protein complex that supports RNA interference, which is at least related to the miRNA pathway in cells. The inventors have found that the judicious use of mismatches in the complementary RNA strand significantly enhances the activity of said miRNA.
Further definitions for nucleic acids, nucleobases, nucleosides, nucleotides, nucleic acid analogues, modified nucleotides, preparation of nucleic acids, design of miRNAs, 5′ blocking agents, host cells and target cells, delivery methods, and nanoparticle functionalisation are preferably as described in WO2013/095132.
Therapeutic Applications
miRNAs that affect phenotypic traits provide intervention points for therapeutic applications as well as diagnostic applications (by screening for the presence or absence of a particular miRNA, or altered concentration of a particular miRNA). It is specifically contemplated that RNA molecules of the present invention can be used to treat any of the diseases or conditions discussed in the previous section. Moreover, any of the methods described above can also be employed with respect to therapeutic and diagnostic aspects of the invention. For example, methods with respect to detecting miRNAs or screening for them can also be employed in a diagnostic context. In therapeutic applications, an effective amount of the miRNAs of the present invention is administered to a cell, which may or may not be in an animal. In some embodiments, a therapeutically effective amount of the miRNAs of the present invention is administered to an individual for the treatment of disease or condition. The term “effective amount” as used herein is defined as the amount of the molecules of the present invention that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term “therapeutically effective amount” as used herein is defined as the amount of the molecules of the present invention that achieves a desired effect with respect to a disease or condition associated with a disease or condition as earlier defined herein. A skilled artisan readily recognizes that in many cases the molecules may not provide a cure but may provide a partial benefit, such as alleviation or improvement of at least one symptom. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of molecules that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount.”
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise 2% to 75% of the weight of the unit, or 25% to 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise less than 1 microgram/kg/body weight, or 1 microgram/kg/body weight, from 5 microgram/kg/body weight, 10 microgram/kg/body weight, 50 microgram/kg/body weight, 100 microgram/kg/body weight, 200 microgram/kg/body weight, 350 microgram/kg/body weight, 500 microgram/kg/body weight, 1 milligram/kg/body weight, 5 milligram/kg/body weight, 10 milligram/kg/body weight, 50 milligram/kg/body weight, 100 milligram/kg/body weight, 200 milligram/kg/body weight, 350 milligram/kg/body weight, or 500 milligram/kg/body weight, to 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of 5 mg/kg/body weight to 100 mg/kg/body weight, 5 microgram/kg/body weight to 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens, chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
The molecules may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
The composition is generally a suspension of nanoparticles in an aqueous medium. However, it can be lyophilized and provided as a powder, wherein the powder comprises the nanoparticles and optionally buffer salts or other excipients.
Effective Dosages
The molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the molecules of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent the symptoms, or prolong the survival of the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. Dosage amount and interval may be adjusted individually to provide plasma levels of the molecules which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from 0.01 to 0.1 mg/kg/day, or from 0.1 to 5 mg/kg/day, preferably from 0.5 to 1 mg/kg/day or more. Therapeutically effective serum levels may be achieved by administering multiple doses each day.
In cases of local administration or selective uptake, the effective local concentration of the proteins may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. The amount of molecules administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. The therapy may be repeated intermittently while symptoms detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs or treatment (including surgery).
Sequence Identity
“Sequence identity” is herein defined as a relationship between two or more nucleic acid (nucleotide, polynucleotide, RNA, DNA) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). In an embodiment, identity is assessed on a whole length of a given SEQ ID NO.
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.
Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.
Chemotherapeutic Agents
Examples of chemotherapeutic agents for use in combinations according to the invention include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholmo-doxorubicm, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, δ-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, δ-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; δ-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-II); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; gefitinib and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumours such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYI 17018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-α, Raf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above. A list of U.S. FDA approved oncology drags with their approved indications can be found on the World Wide Web at accessdata.fda.gov/scripts/cder/onctools/druglist.cfm. A suitable RNR inhibitor is selected from the group consisting of gemcitabine, hydroxyurea, clolar, clofarabine, and triapine. A suitable AURKB inhibitor is selected from the group consisting of: AZD1152, VX-680, MLN8054, MLN8237, PHA680632, PH739358, Hesperidin, ZM447439, JNJ770621, SU6668, CCT129202, AT9283, MP529, SNS314, R763, ENMD2076, XL228, TTP687, PF03814735 and CYC116. Another suitable anticancer drug is gefitinib.

FIGURE LEGENDS

FIG. 1 —Canonical pathway analysis. (A) Top 25 canonical pathways regulated by miR-193a in at least three cell lines at 24 h ranked based on P value. Dotted line indicates P<0.01. White bar: activated, black bar: inhibited, grey bar: direction not determined. (B) Treemap of genes that were identified in at least 4 significant pathways. Box size corresponds to the number of pathways in which the gene was differentially expressed.

FIG. 2 —Genes downregulated by miR-193a in the PTEN pathway. Genes that were significantly downregulated by miR-193a (average relative expression compared to mock <1, P<0.05) at 24 h in at least three cell lines are shown without hatching. PTEN is highlighted in black. Pointy arrows indicate stimulation whereas bar-headed arrows indicate inhibition.

FIG. 3 —Biological functions affected by miRNA-193a. Biological functions relevant to (tumour) cells with z-scores <−2 and >2. All P values are smaller than 0.00001.

FIG. 4 —Western blotting of miR-193a-3p targets in the PTEN pathway. Human tumor cell lines were transfected with 10 nM scrambled control or 10 nM miRNA-193a and lysed after 72 h. Clarified whole cell lysates were immunoblotted for FAK, P70S6K, PIK3R1 and TGFBRIII. Vinculin and tubulin are loading controls. Boxes indicate protein downregulation.

FIG. 5 —Western blotting of phosphoproteins in the PTEN pathway. Human tumor cell lines were transfected with 10 nM scrambled control or 10 nM miRNA-193a and lysed after 72 h. Clarified whole cell lysates were immunoblotted for pSer473 AKT, AKT, pThr202/Tyr204 ERK1/2, ERK1/2, pSer259 c-RAF and c-RAF. Vinculin and tubulin are loading controls. Full boxes indicate protein downregulation and dashed boxes indicate protein upregulation.

FIG. 6 —Transfection with miRNA-193a induces surface expression of CRT in A2058, HEP3B, HCT116, and Huh7 cells. (A-B) Graphs show percentages of live (DAPI⁻) and dying (DAPI^low), but not dead (DAPI⁺) A2058 and Hep3B cells, expressing CRT on their surface. Cells transfected with 0.1, 1, 3 and 10 nM of miRNA-193a, or a mock transfection control. (C-D) Panels show the cytofluorometric plots of A2058 (C) and HEP3B (D) cells, analyzed 72 hours post transfection. (E-F) Graphs show percentage of live (DAPI⁻) HCT116 and Huh7 cells expressing CRT on their surface, 96 h post transfection with 1 and 10 nM of miRNA-193a, or a mock transfection control. (G-H) Panels show the cytofluorometric plots of HCT116 (G) and Huh7 (H) cells, analyzed 96 hours post transfection.

FIG. 7 —miRNA-193a-transfected cells are able to markedly stimulate maturation of dendritic cells from monocytes in co-culture assay. Monocytes were kept in culture co-culture with mock transfected or miRNA-193a transfected (1 or 10 nM) A2058 cells. Surface expression of CD80 and MHC class II molecules as markers of mature DC, were measured with flow cytometry, 7 days post co-culture. TNFα was used as a known DC maturation stimulator.

FIG. 8 —Co-culture with miRNA-193a transfected A2058 tumor cells enhances the proliferation of T cells. PBMCs were labeled with CFSE and kept in culture alone, or in co-culture with mock transfected or miRNA-193a transfected A2058 cells. Cytofluorometric plots show the level of CFSE of CD3+ T cells, after 2 days or 6 days of co-culture.

FIG. 9 —Effect of human Peripheral Blood Mononuclear Cells (PBMCs) on human melanoma A2058 and NSCLC A549 tumor cells. Human melanoma A2058 (A) and NSCLC A549 (B) tumor cells were co-cultured with the indicated ratio of human PBMCs to tumor cells for 72 h either in the absence or the presence of human anti CD3/CD28 antibodies (T cell activator). Then surviving cells were fixed and stained with crystal violet. The relative percentage of surviving cells (as compared to similar experimental conditions in the absence of PBMCs) was quantified by colorimetry of the stained cells (Feoktistova et al., 2016). Error bars represent SD to the mean of 3 independent replicates.

FIG. 10 —Effect of human Peripheral Blood Mononuclear Cells (PBMCs) on human melanoma A2058 (A) and NSCLC A549 (B) tumor cells upon tumor cell transfection with miRNA-193a. Human melanoma A2058 and NSCLC A549 tumor cells were transfected (RNAiMAX) with the indicated concentrations of either negative miRNA control or miR-193a-3p, after which cells were co-cultured with the indicated ratio of human PBMCs to tumor cells for the indicated times. Then surviving cells were fixed and stained with crystal violet. The relative percentage of surviving cells (as compared to mock transfected condition)) was quantified by colorimetry of the stained cells (Feoktistova et al., 2016). N.S.: not significant, *: p<0.05 and **: p<0.01 as determined by the Student t tests (asymptotic significance [2-tailed]). Error bars represent SD to the mean of 3 independent replicates.

EXAMPLES

Example 1—RNA-Sequencing, Differential Gene Expression, and Pathway Analysis after Treatment of Different Cancer Cell Lines with miRNA-193a

Implementation of high-throughput RNA-sequencing (RNA-seq) has become a powerful tool for comprehensive characterization of the whole transcriptome at gene and exon levels and with a unique ability to identify differentially expressed genes, novel genes and transcripts at high resolution and efficiency. However, till date, very few miRNAs have been characterized for their specific role in cancer development. Hence, we have used the high-throughput RNA-seq after overexpressing a miRNA-193a (viz. a miRNA-193a-3p mimic) in 5 different cancer cell lines including A540 and H460 (both lung cancer), Huh7 and Hep3B (both liver cancer), and BT549 (breast cancer) at 24 h post-transfection with miR-193a at 10 nM. The gene expression was compared to mock as control and differentially expressed genes and their cellular pathways were subsequently identified.

1.1 Materials & Methods

1.1.1 Cell Preparation for RNA-Sect
Human cancer cell lines were cultured in appropriate media (Table 1) and seeded into δ-well plates 24 h before transfection with 10 nM miRNA-193a-3p mimic or mock using Lipofectamine RNAiMAX (Thermofisher). The mimic was a double stranded mimic wherein the antisense strand consisted of an RNA oligonucleotide having SEQ ID NO: 56 (the canonical miRNA-193a-3p), and wherein the sense strand consisted of an oligonucleotide represented by SEQ ID NO: 218.
Reagents were aspirated 16 h after transfection and cells were passaged into new δ-well plates. Media was aspirated 24 h after transfection and plates were stored at −80° C. Three independent replicates were performed for each cell line.

TABLE 1

Cell line details.

Cell line	Cancer type	Medium

A549	Lung (NSCLC)	F-12K + 10% FBS + P/S
BT549	Breast (TNBC)	RPMI-1640 + 10% FBS + P/S +
		0.023 IU/mL insulin
H460	Lung (NSCLC)	RPMI-1640 + 10% FBS + P/S
HEP3B	Liver (HCC)	EMEM + 10% FBS + P/S
HUH7	Liver (HCC)	DMEM low glucose + 10% FBS +
		P/S + L-glutamine

FBS: fetal bovine serum,
P/S: penicillin streptomycin

1.1.2 RNA Isolation for RNA-Sect
RNA was isolated using the miRNeasy Mini kit (Qiagen). The procedure included on-column DNase treatment. RNA concentration was measured on NanodropOne. 150 ng of each independent replicate was pooled and 450 ng samples (having sample IDs A549 Mock_24, A549 miRNA-193a-3p_24, BT549 Mock_24, BT549 miRNA-193a-3p_24, H460 Mock_24, H460 miRNA-193a-3p_24, HEP3B Mock_24, HEP3B miRNA-193a-3p_24, HUH7 Mock_24, and HUH7 miRNA-193a-3p_24) were submitted to GenomeScan BV (Leiden, The Netherlands).
1.1.3 RNA-Seq Procedure
PolyA enrichment was performed followed by next generation RNA-Seq using Illumina NovaSeq 6000 at GenomeScan BV. The data processing workflow included raw data quality control, adapter trimming, and alignment of short reads. The reference GRCh37.75.dna.primary_assembly was used for alignment of the reads for each sample. Based on the mapped locations in the alignment file the frequency of how often a read was mapped on a transcript was determined (feature counting). The counts were saved to count files, which serve as input for downstream RNA-Seq differential expression analysis.
1.1.4 Data Analysis for RNA-Sect
Differential expression analysis was performed on the short read data set by GenomeScan BV. The read counts were loaded into the DESeq package v1.30.0, a statistical package within the R platform v3.4.4. DESeq was specifically developed to find differentially expressed genes between two conditions (mock versus miRNA-193a-3p) for RNA-seq data with small sample size and over-dispersion. The differential expression comparison grouping is provided in Table.

TABLE 2

Expression comparison setup.

Comparison	Condition A	Condition B

1	A549_Mock_24	A549_miRNA-193a-3p_24
2	BT549_Mock_24	BT549_miRNA-193a-3p_24
3	H460_Mock_24	H460_miRNA-193a-3p_24
4	HEP3B_Mock_24	HEP3B_miRNA-193a-3p_24
5	HUH7_Mock_24	HUH7_miRNA-193a-3p_24

1.1.5 Pathway Analysis
Lists of genes that were significantly (P<0.05) differentially expressed in our RNA-seq dataset were uploaded and analyzed using Ingenuity Pathway Analysis (IPA) software (www.ingenuity.com).

1.2 Results

1.2.1 Genes Regulated by miR-193a-3p Mimic in Solid Tumor Cell Lines
Lists of significantly (P<0.05) differentially expressed genes (relative expression miRNA-193a/relative expression mock) at 24 h after transfection were created for all cell lines (see description of invention). Most genes were downregulated as compared to mock (relative expression miRNA-193a/relative expression mock <1) (see Table 3).

TABLE 3

Number of genes down- and upregulated
by 193a-3p mimic per cell line.

24 h

	Down	Up

A549	615	220
BT549	620	168
H460	656	215
HEP3B	599	166
HUH7	683	200

Table 4 shows genes with known roles in cancer that were downregulated by miRNA-193a in each cell line. Genes that were downregulated in all cell lines include: CCND1, CDK6, KRAS, MCL1, NTSE, STMN1, TGFBR3 and YWHAZ.

TABLE 4

Genes of interest downregulated by miR-193a per cell line.

Cel lines	Downregulated genes

A549	CAPRIN1, CCNA2, CCND1, CDK4, CDK6,
	CHEK1, DCAF7, DDB1, ETS1, HDAC3,
	HMGB1, IL17RD, KRAS, MCL, MPP2,
	NOTCH2, NT5E, PLAU, PSEN1, PTK2,
	RAB27B, SEPN1, SLC7A5, SOS2, ST3GAL4,
	STAT3, STMN1, TGFB2, TGFBR2, TGFBR3,
	TNFRSF21, YAP1, YWHAZ
BT549	CCNA2, CCND1, CDC25A, CDK4, CDK6,
	CSF2, DCAF7, DDB1, ETS1, GRB7,
	HIC2, IDO1, IL17RD, KRAS, MCL1,
	MDM2, MPP2, NOTCH2, NT5E, PLAU,
	PSEN1, PTK2, RAB27B, SEPN1, SLC7A5,
	SOS2, ST3GAL4, STMN1, TGFBR3,
	TNFRSF1B, TNFRSF21, YAP1, YWHAZ
H460	CAPRIN1, CCNA2, CCND1, CDK6, CDKN1A,
	CHEK1, CXCL1, CXCL5, DCAF7, DDB1,
	ETS1, HMGB1, IL17RD, KRAS, MAPK8,
	MCL1, MPP2, NOTCH1, NOTCH2, NOTCH3,
	NT5E, PLAU, PSEN1, PTK2, SEPN1,
	SLC7A5, ST3GAL4, STMN1, TGFBR3,
	TNFRSF1B, TNFRSF21
HEP3B	AJUBA, CAPRIN1, CCND1, CDK6, CRYAA,
	DCAF7, ERBB4, ETS1, GRB7, IL17RD,
	KRAS, MAPK8, MCL1, MDM2, MPP2, NOTCH1,
	NT5E, PSEN1, PTK2, SEPN1, SLC7A5,
	SOS2, ST3GAL4, STMN1, TGFBR2, TGFBR3,
	TNFRSF1B, TNFRSF21, YAP1, YWHAZ
HUH7	BRCA1, CCNA2, CCND1, CDC25A, CDK1,
	CDK6, CHEK1, DCAF7, E2F1, ETS1, EZH2,
	FEN1, FOXM1, GRB7, HMGB1, IL17RD,
	KRAS, MAPK8, MCL1, MDM2, MELK, MPP2,
	NT5E, PLAU, PLK1, RAD51, SEPN1, SLC7A5,
	ST3GAL4, STMN1, TGFBR3, TNFRSF21, YWHAZ

1.2.2 Cellular Pathways Regulated by miR-193a in Solid Tumor Cell Lines
IPA was performed to identify canonical pathways that are affected in miRNA-193a treated cells compared to mock, based on the differential expression data. Because the objective was to develop new treatment options by more closely defining the mode of action of miR-193a across cancer types, we next analyzed the pathways that were regulated by genes differentially expressed in at least three cell lines. This analysis showed that the majority of pathways was affected or inhibited (FIG. 1A), including many growth factor signalling pathways which induce cellular proliferation and tumour progression. The most enriched canonical pathway, the tumour suppressive PTEN pathway, was indicated to be activated (z-score of 2.309). Differentially expressed genes in this pathway include RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, and MAGI3 (FIG. 2).
Other identified pathways were significantly inhibited, including Neuregulin signalling (z-score of −2.333) and HGF signalling (z-score of −3.162). Genes from our differential expression dataset that participate in these pathways are shown in FIG. 1 b and include PI3KR1, KRAS, SOS2 and PTK2. Many are important components of growth factor signalling and mitogen-activated protein kinase (MAPK) pathways, inducing nuclear signals for cellular proliferation and tumour progression.
Subsequently, IPA software was used to predict downstream effects of the observed gene expression changes on biological functions and disease processes. Out of the 100 most significant biological functions that were changed by 193a at 24 h, those that were inhibited (z-score <−2) were related to cell survival, proliferation, migration, or cancer, and those that were activated (z-score >2) were related to (tumour) cell death (FIG. 3 ). Furthermore, the majority of the affected biological functions (55% at 24 h) belonged to the category Cancer (Table 5, which shows categories of top 100 biological functions ranked based on the number of miRNA-193a-regulated functions (all P<0.00001) at 24 h).

TABLE 5

Biological function categories.
24 h

	Category	# functions

	Cancer	55
	Cell movement	10
	Cell death and survival	9
	Cell growth and development	7
	Organismal development and survival	6
	Nervous system function	5
	Cardiovascular	3
	Cell maintenance	3
	DNA replication	1
	Hematological disease	1

Example 2—RNA-Sequencing, Differential Gene Expression, and Pathway Analysis after Treatment of Different Cancer Cell Lines with miRNA-193a

miRNA-193a was tested in different cancer cell lines (see Table 2.1). The cells were treated with miRNA-193a as described for example 1 at different concentrations (1, 3, 10 nM). Controls (mock, untreated, and scrambled) were measured for all cell types. Assays were performed after 24 h, 48 h and 72 h. Table 2.1 shows results at 10 nM concentration at indicated time points. The results were quantified and normalized to the mock control. 10 nM was a suitable concentration, because the cells showed no signs of a toxic effect at that concentration.

TABLE 2.1

effect of miRN-193a on various tambours

				Cell cycle
Cancer	Cell	Viability	Apoptosis	arrest	Motility
type	line	(96 h)	(48/72 h)	(72 h)	(18 to 24 h)

Liver	HEP3B	<50%	<2x	G2/M	>50%
	HUH7	<50%	<2x	—	n.a.
Lung	A549	<50%	>2x	SubG1	>50%
	H460	<50%	>2x	SubG1	n.a

miRNA-193a treatment in the cancer cell lines decreased cell viability over time as measured by either an MTS assay or by cell count. Apoptosis induction was enhanced over time as measured by a caspase 3/7 apoptosis assay. Cell cycle arrest profiles were measured performing either nuclei imaging or flow cytometry. miRNA-193a treatment induced either a G2/M or a SubG1 cell cycle arrest profile in a manner depending on the cell line. While in HUH7 no obvious cell cycle arrest profile was observed following the indicated methods, an increased apoptosis was observed indicated by Caspase 3/7 activation and enhanced cleaved-parp protein on western blot (data not shown) following miRNA-193a treatment in this cell line. This result indicates that miRNA-193a treatment affects the viability of the cells. Cell motility of two cell lines was significantly decreased after treatment as assessed via a Boyden chamber assay.

Conclusion

miRNA-193a treatment decreased cell viability partly by inducing apoptosis and by an increase in the cell cycle arrest profile. miRNA-193a treatment also decreases cell motility of cancer cells, indicating its role in the inhibition of cancer cell migration.

Example 3— Further Study of the PTEN Pathway Activation

Example 1 shows that the IPA analysis identified the tumor suppressive PTEN pathway as the most enriched canonical pathway which was activated by miRNA-193a. Here regulation of selected miRNA-193a targets is analysed at the protein level by western blotting, including: FAK (PTK2), P70S6 (RPS6KB2), PI3KR1, TGFBRIII and other important signaling molecules including P-AKT, AKT, p-ERK1/2, ERK1/2, p-c-RAF and c-RAF, all factors in the PTEN pathway.

Materials and Methods

Cell Preparation

Human cancer cell lines were cultured in appropriate media (see table below) and seeded into δ-well plates before transfection with 10 nM miRNA-193a-3p mimic as described in example 1, 10 nM scrambled random control, or mock using Lipofectamine RNAiMAX (Thermofisher). Media was aspirated 72 h after transfection and plates were stored at −80° C.

3. 1. Cell Lines Details


Cell
line	Cancer type	Medium

A549	Lung (NSCLC)	F-12K + 10% FBS + P/S
BT549	Breast (TNBC)	RPMI-1640 + 10% FBS + P/S +
		0.023 IU/mL insulin
H460	Lung (NSCLC)	RPMI-1640 + 10% FBS + P/S
HEP3B	Liver (HCC)	EMEM + 10% FBS + P/S
HUH7	Liver (HCC)	DMEM low glucose + 10% FBS +
		P/S + L-glutamine
PANC-1	Pancreas	DMEM + 10% FBS + P/S
SNU449	Liver (HCC)	RPMI-1640 + 10% FBS + P/S

FBS: fetal bovine serum,
P/S: penicillin streptomycin

Protein Isolation and Quantification

RIPA buffer (50 mM Tris- HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM EDTA), supplemented with protease and phosphatase inhibitor cocktails, was added to harvested cells. Lysates were centrifugated at 15,000 g for 1 h at 4° C. and clarified by removing the cell debris pellet. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher).

Electrophoresis and Immunoblotting

Samples were separated at 120 V by SDS-PAGE on Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad). Proteins were transferred at 200 mA for 2 h to PVDF membranes in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membranes were blocked using 5% milk or 5% BSA in Tris-buffered saline with Tween (20 mM Tris pH 7.6, 137 mM NaCl, 0.1% Tween). Blots were probed with primary and horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using ECL reagents. Membranes were stripped in stripping buffer (62.5 mM Tris pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol) for 30 min at 50° C. and reprobed as appropriate.

Results

Lysates from cells transfected with 10 nM scrambled control or 10 nM miRNA-193a-3p mimic as described in example 1 were immunoblotted to assess the protein level of selected predicted miR-193a-3p target genes as well as phosphorylation status of key signalling proteins in the PTEN pathway. In all tested cell lines (A549, HUH7, SNU449, BT549, H460, A2058, HEP3B and PANC-1) downregulation of FAK, also called PTK2, was observed in the miRNA-193a sample compared to mock and scrambled control (FIG. 4 ). TGFBRIII was also downregulated by miRNA-193a in cell lines where a constitutive expression level could be observed (A549, HUH7, SNU449, BT549 and H460). Protein level of PIK3R1, the regulatory subunit of PI3K, was decreased in all cell lines except SNU449. P70S6, also called RPS6KB2, was downregulated in H460, A2058 and HEP3B. Vinculin and tubulin were used as loading controls. In A549 and H460, tubulin was affected by miRNA-193a, whereas vinculin was stable, indicating that miRNA-193a does not reduce general protein levels. Additionally, we observed downregulation of AKT phosphorylation by miRNA-193a in most cell lines (A549, A2058, SNU449, HUH7, H460 and HEP3B (FIG. 5 ). Interestingly, miRNA-193a increased phosphorylation of ERK in at least two cell lines (A549 and A2058). In SNU449, both ERK phosphorylation and ERK total protein level were upregulated. Phosphorylation of c-RAF was only downregulated in PANC-1.

Conclusion

These results are in line with the RNA-sequencing data obtained previously. miRNA-193a-3p mimic miRNA-193a decreased protein expression of FAK, P70S6K, PIK3R1 and TGFBRIII in multiple human tumor cell lines. In addition, treatment of cells with miRNA-193a-3p mimic miRNA-193a lead to reduced phosphorylation of AKT, which could be due to downregulation of upstream signaling proteins such as PIK3R1 and FAK. Furthermore, we observed increased phosphorylation of ERK, which could be a consequence of decreased AKT activity via effects on RAF, although phosphorylation of c-RAF was decreased in only one cell line (PANC-1). Increased phosphorylation of ERK may also be the result of other upstream events, including decreased phosphatase activity or increased activity of upstream kinases.

Example 4— miRNA-193a is an Immunogenic Cell Death (ICD) Inducer

Introduction: The concept of Immunogenic Cell Death (ICD) has been defined as a unique class of regulated cell death capable of eliciting antigen-specific adaptive immune responses through the emission of a spatiotemporally defined set of danger signals known as damage associated molecular patterns (DAMPs) (Krysko et al., Nat. Rev. Cancer, 2012; Casares et al., J. Exp. Med., 2005; Kroemer et al., Annu. Rev. Immuna, 2013). The most notable DAMPs are: release of HGMB1, release of ATP and surface expression of calreticulin (CRT), as a sign of ER stress. Induction of ICD by some (specific) anticancer agents upon induction of cancer cell death leads to release of DAMPs into the tumor microenvironment (TME), which operate on receptors expressed by dendritic cells (DCs) to accelerate their maturation, and in turn stimulate presentation of tumor-associated antigens to T cells, leading to T cell activation and proliferation eventually culminating in enhanced cytotoxicity against the tumor cells, and formation of an immunological memory against the tumor antigens.

Materials and Methods

Transfection: A2058 melanoma, HEP3B and Huh7 hepatocyte, and HCT116 colon tumor cells were transfected with different concentrations of miRNA-193a as described in example 1, or a mock (“fake transfection”) control. In brief, 5×10⁵A2058 or HEP3B cells were seeded in 1.5 mL complete media in δ-well cell culture plates. Both cell lines were transfected 4 h later. A 500 μL transfection mix containing 7.5 μL Lipofectamine RNAiMAX (Thermo Fisher) and the appropriate concentration miRNA-193a-3p was added to each well. Transfection conditions included were 0.1, 1, 3 or 10 nM miRNA-193a and the mock-transfected negative control. Huh7 and HCT116 cell lines were transfected 24 h later. First the media was replaced by 1.5 mL of fresh media. Then, a 500 μL transfection mix containing 7.5 μL Lipofectamine RNAiMAX and the appropriate concentration of miRNA-193a was added to each well. Transfection conditions included were 1 or 10 nM miRNA-193a and the mock-transfected negative control. All cell lines were passaged into 24-well plates 16 h after transfection by aspirating and retaining media in 5-mL tubes, washing 1× with TrypLE (Gibco), and incubating for 10 to 12 min until detached. Cells were collected with 1 mL fresh media and added to the retained media. Tubes were centrifuged for 5 min at 1,500 RPM and supernatant removed. Cells were resuspended in 500 μL fresh media and counted using a 1:1 dilution with trypan blue using the Luna-II cell counter (Westburg). 5×10⁴cells in 1 mL fresh media were seeded per well.
Flow cytometry: For flow cytometric analysis at mentioned time post transfection, cells were harvested after washing 1× with TrypLE (Gibco), and incubating for 10 to 12 min until detached. For each condition, 200 μL of single cell suspensions containing 5×10⁴cells were prepared in 4-mL polypropylene tubes. Cells were stained with fluorescently labeled antibodies in a 1:200 dilution. The expression of CRT was measured using a DyLight™ 488 conjugate anti-human Calreticulin (CRT) antibody (Clone FMC 75, Enzo Life science). To detect the maturation state of DCs, the surface expression of CD80, and MHC class II molecules were measured using PerCP/Cyanine5.5 anti-human CD80 antibody (Clone 2D10, BioLegend), and APC anti-human HLA-DR, DP, DQ antibody (Clone Tü39, BioLegend), respectively. Also, DAPI (BioLegend) was added at a final concentration of 2 μM, to detect live/dead cells, and dead cells were excluded from further analyses. Flow cytometry was performed using a FACSCanto II cytometer (BD Biosciences), data was analyzed with FlowJo software (Tree Star inc.).
Co-culture with CFSE labeled PBMCs: PBMCs were isolated from fresh blood buffy coat (Sanquin), using SepMate™-50 tubes (STEMCELL), following manufacturer's protocol. Ficoll® Paque Plus (SigmaAldrich) was used as the density gradient medium. PBMCs were then labeled with CFSE using CFSE Cell Division Tracker Kit (BioLegend), following the manufacturer's protocol. A2058 cells were transfected and 16 h after transfection, cells were passaged to a 24 well plate as explained before. 3×10⁴A2058 cells were seeded in 0.5 mL of fresh medium into each well. Also, 0.5 mL of CFSE labeled PBMC suspensions containing 1.2×10⁵PBMCs was added into each well. Same amount of PBMCs, without any A2058 cells, was cultured as “PBMC only” control condition. The co-culture was incubated at 37° C. for mentioned time. For detection of T cells, cells were stained with Brilliant Violet 510™ anti-human CD3 Antibody (Clone UCHT1, BioLegend) in a 1:200 dilution.
Isolation of monocyte from PBMC: Monocytes were isolated from PBMCs using MACS cell-separation system and pan monocyte isolation kit (Miltenyi Biotec), according to the manufacturer's protocol. In brief, PBMCs were labeled with a cocktail of biotin-conjugated monoclonal antibodies against antigens expressed on all different main cell types present among the PBMCs, except monocytes. Labeled cells then passed through columns containing micro beads conjugated to monoclonal anti-biotin antibodies. This approach enabled isolation of highly pure unlabeled and intact monocytes, by depletion of the magnetically labeled non-monocyte cells.
Co-culture of A2058 cells with monocytes: A2058 cells were transfected with 1 or 10 nM concentrations of miRNA-193a, or a mock-transfected negative control, as explained earlier. Cells were harvested 16 h after transfection, as explained before, and seeded into 96-well plates. 4×10³cells in 100 μL fresh media were seeded per well. 4 hours later, 100 μL of DC generation medium (PromoCell), containing 4×10³-, 4×10⁴-, or no-monocytes (depending on the “monocyte:tumor cell ratio” condition) was added into each well. 4 days after starting of co-culture, the medium in all the wells were replaced with 150 μL of fresh DC generation medium. To have a positive control for mature DCs, 6 day after putting cells into the co-culture, component B of DC generation cytokine pack (C-28050, PromoCell) containing TNFα as a DC-maturation stimulator, was added to the wells with positive control condition (1.5 μL/well of component B cytokine pack, to have a final TNFα concentration of 5000 U/mL). On day 7 post co-culture, cells were stained with fluorescent antibodies against cell surface markers of mature DC, and analyzed with flow cytometry, as explained earlier.

Results

To investigate the effect of miRNA-193a on tumor cells, the expression of CRT on the surface of miRNA-193a transfected tumor cells was assessed by flow cytometry. As shown in FIG. 6 , miRNA-193a induced expression of the CRT marker on the cell surface in A2058 cells (up to 46% after 72 h) (FIGS. 6 -A and C), and in HCT116 cells (up to 65% after 96 h) (FIGS. 6 -E and G), compared with mock transfected cells containing only 5% and 10% surface-CRT′ cells, respectively. The induction of CRT marker was also observed to a lesser extent in Hep3B cells (up to 8% after 72 h) (FIGS. 6 -B and D), and in Huh7 cells (up to 35% after 96 h) (FIGS. 6 -F and H), as compared to mock transfected cells containing only 4% and 23% surface-CRT′ cells, respectively. Moreover, via targeting two major ectonucleotidases CD39 and CD73, miRNA-193a can prevent the conversion of extracellular ATP to ADP, AMP and adenosine, and thereby retains the ATP content of the TME.
Next, we investigated whether the cytotoxic effect of miRNA-193a on A2058 cells, can stimulate the maturation process of monocytes towards full mature DCs. To address this, monocytes were put in co-culture with miRNA-193a- or mock-transfected A2058 cells. 7 days post co-culture, the frequency of cells expressing mature DC surface-markers (CD80 and MHC II) were measured with flow cytometric analysis. To make a positive control, TNFα was used as a known DC maturation inducer. As shown in FIG. 7 , when monocytes were co-cultured with 10 nM miRNA-193a transfected A2058 cells in a 10 to 1 ratio, the frequency of CD80⁺ and MHC II⁺ cells were significantly increased (10× and 4.9×, respectively), compared to co-culture with mock transfected cells. These results indicate that miRNA-193a-transfected A2058 cells are able to markedly stimulate maturation of dendritic cells from monocytes in cell-based assays.
Finally, we addressed the effect of miRNA-193a on proliferation of T cells in co-culture with miRNA-193a transfected tumor cells. PBMCs were labeled with CFSE, a fluorescent non-toxic marker that can be retained within the cells and gets diluted with each cell division. Levels of CFSE measured by flow cytometry were compared between three conditions: 1) PBMCs in culture alone, 2) PBMCs in culture with mock transfected A2058 cells, and 3) PBMCs in culture with miRNA-193a 1 nM transfected A2058 cells. The results show that keeping PBMCs in co-culture with miRNA-193a-transfected A2058 cells enhanced the proliferation of T cells (FIG. 8 ).
Furthermore, miRNA-193a increased the vulnerability of tumor cells to PBMC-mediated cytotoxicity, as showed by fixation, staining and colorimetric quantification of survived tumor cells following co-culture with PBMCs. Interestingly, in vivo experiments in a syngeneic murine 4T1 orthotopic breast cancer model confirmed the formation of a long-term T cell mediated anti-tumor immunity in miRNA-193a treated animals, or in naïve mice that had received an adoptive T cell transfer from miRNA-193a treated mice.
Taken together, these results strongly suggest that miRNA-193a is a bona fide ICD inducer which kills the tumor cells in a way that not only stimulates PBMC-mediated cytotoxicity to enhance overall anti-tumor efficacy, but also stimulates DC maturation and activates the formation of an adaptive anti-tumor immunity.

Example 5 Effect of miRNA-193a on Human PBMC-Mediated Tumor Cell Killing Following Transfection in Human Tumor Cells

One of the most recent developments in the understanding of cancer biology is the field of immuno-oncology (10). Often tumors present the ability to evade cancer immunosurveillance, which represents one of the hallmarks of cancer (Hanahan et al., 2011) Accordingly, the main goals of cancer immunotherapy are to strengthen the patient's immune response to the tumor by improving its capacity for tumor recognition and the disruption of immunosuppressive mechanisms (Chen et al., 2017). As part of the induction mechanisms supporting pronounced immune suppression of the tumors, adenosine levels in the tumor microenvironment (TME) have recently attracted significant attention to develop novel therapeutic intervention in oncology. Adenosine in the tumor microenvironment (TME) is generated mainly by ectonucleotidases CD39 (ENTPD1; which converts extracellular adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and then to adenosine monophosphate (AMP)) and CD73 (NT5E; which is responsible for the generation of adenosine from AMP) (Stagg et al., 2010). NT5E can act as inhibitory immune checkpoint molecule, since free adenosine generated by NT5E inhibits cellular immune responses, thereby promoting immune escape of tumor cells. Indeed, adenosine is a potent immunosuppressive metabolite that is generated in response to pro-inflammatory stimuli, such as cellular stress initiated by hypoxia or ischemia. Landmark studies by Ohta and colleagues have highlighted the importance of adenosine for tumor immune escape (Ohta et al., 2006). Extracellular adenosine concentrations in solid tumors are reported to be higher than under normal physiological conditions (Blay et al., 1997).
Our transcriptome analysis identified a pool of immune related genes among the genes whose expression was affected by a mimic of miR-193a-3p as described in example 1. Among them were modifiers of TME, such as CD73. Moreover, our in vivo studies in murine models strongly suggested that miR-193a-3p, on top of its other effects, modifies the interaction between tumor cells and the immune system in a way that immune cells become more active in killing tumor cells. To assess the 10 related effect of miR-193a-3p in human cells, and also to investigate the mechanism of the miR-193a-3p mediated 10 effect, we established an in vitro assay in which, tumor cells were co-cultured together with human peripheral blood mononuclear cells (hPBMCs) isolated from healthy donor's peripheral blood, and the cytotoxic effect of hPBMCs on tumor cells was assessed with or without transfection with miR-193a-3p (see example 4).
As a first step and to establish the technical validity of such a cell-based assay, human anti CD3/CD28 T cell activator antibodies (positive control) was added to the tumor cells and PBMCs co-culture. The used activator comprises a soluble tetrameric antibody complex that binds CD3 and CD28 immune cell surface ligands. This binding results in cross-linking of CD3 and CD28, thereby providing the required primary and co-stimulatory signals for an effective T cell activation (Riddell et al., 1990; Bashour et al., 2014). As illustrated in FIG. 9 , although unstimulated human PBMCs showed limited effect on tumor cell survival (co-culture), addition of anti CD3/CD28 antibodies in the co-culture led to a pronounced decrease in tumor cell survival, most likely consequent to an efficient T cell activation and subsequent T cell-mediated tumor cell killing. Interestingly, in similar study performed with primary human dermal fibroblasts, no effect of anti CD3/CD28 on fibroblast viability was observed (data not shown), strongly suggesting that experimental T cell activation does not lead to T cell-mediated normal fibroblast killing.
Next, human melanoma A2058 and NSCLC A549 tumor cells were transfected with increasing concentrations of miR-193a-3p after which they were co-cultured with human PBMCs (at different PBMCs:Tumor cells ratio) for different times. Human PBMCs from independent donors were able to induce time-dependent marked tumor cell killing upon transfection of tumor cells with miRNA-193a as described in example 1, but not the (negative) miRNA control (scramble), validating sequence-specificity of miRNA-193a activity (FIG. 10 ).
Taken together, our results demonstrate that transfection of tumor cells with miR-193a-3p clearly increases the vulnerability of tumor cells (e.g., A2058 and A549 tumor cells) to human PBMC cytotoxicity, by sensitizing tumor cells to PBMCs, and/or by releasing signals from transfected tumor cells to activate T cell-containing PBMCs.

Claims

1. A method for treating a condition associated with low expression of calreticulin (CRT) or with an impaired immunogenic cell death (ICD) pathway, the method comprising the step of administering a miRNA-193a or a source thereof, or composition comprising a miRNA-193a or a source thereof.

2. The method according to claim 1, wherein the miRNA-193a is a CRT agonist or promotes cell surface expression of CRT or rescues or restores the ICD pathway.

3. The method according to claim 1, wherein the miRNA-193a is a miRNA-193a molecule, an isomiR, or a mimic thereof.

4. The method according to claim 1, wherein a source of a miRNA is a precursor of a miRNA and is a nucleic acid of at least 50 nucleotides in length.

5. The method according to claim 1,

wherein said miRNA shares at least 70% sequence identity with any one of SEQ ID NOs: 56, 121, or 122,

and/or wherein said miRNA is from 15-30 nucleotides in length,

and/or wherein said source of a miRNA is a precursor of said miRNA and shares at least 70% sequence identity with any one of SEQ ID NOs: 5 or 13.

6. The method according to claim 1, wherein the condition associated with low expression of CRT is a low-CRT cancer or a cancer wherein the ICD pathway is impaired.

7. The method according to claim 1, wherein the low-CRT cancer is a low-CRT sarcoma, brain cancer, head and neck cancer, breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, ovarian cancer, melanoma, pancreatic cancer, thyroid cancer, hamartoma, tumour of the haematopoietic and lymphoid malignancy, or prostate cancer.

8. The method according to any one of claims 1-7, wherein the miRNA-193a modulates expression of a gene selected from the group consisting of CRT, RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1, and HMGB1.

9. (canceled)

10. The method according to claim 1, wherein the composition further comprises a further miRNA or precursor thereof, wherein the further miRNA is selected from the group consisting of miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or an isomiR thereof, or a mimic thereof.

11. The method according to claim 1, wherein the composition further comprises an additional pharmaceutically active compound.

12. The method according to claim 1, wherein the composition is a nanoparticle composition, the nanoparticle comprising a diamino lipid and the miRNA-193a or a source thereof, wherein the diamino lipid is of general formula (I)

wherein

n is 0, 1, or 2, and

T¹, T², and T³are each independently a C₁₀-C₁₈chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C₁-C₄alkyl, C₁-C₄alkenyl, and C₁-C₄alkoxy.

13. The method according to claim 12, wherein the nanoparticles comprises:

i) 20-60 mol % of diamino lipid, and

ii) 0-40 mol % of a phospholipid, and

iii) 30-70 mol % of a sterol, and

iv) 0-10 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.

14. An in vivo, in vitro, or ex vivo method for agonising CRT or for increasing cell surface expression of CRT, the method comprising the step of contacting a cell with a miRNA-193a or a source thereof, or with a composition comprising a miRNA-193a or a source thereof.

15. A method for treating a low-CRT cancer, the method comprising the step of administering to a subject a miRNA-193a or a source thereof, or a composition comprising a miRNA-193a or a source thereof.

16. The method according to claim 1, wherein the miRNA-193a is an oligonucleotide with a seed sequence comprising at least 6 of the 7 nucleotides of the seed sequence represented by SEQ ID NO: 22.

17. The method according to claim 8, wherein the miRNA-193a modulates expression of a gene selected from the group consisting of RPS6KB2, KRAS, PDGFRB, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1.

18. The method according to claim 8, wherein the miRNA-193a modulates expression of a gene selected from PDPK1 and INPPL1.

19. The method according to claim 11, wherein the additional pharmaceutically active compound is selected from the group consisting of a PP2A methylating agent, an inhibitor of hepatocyte growth factor (HGF), an antibody, a PI3K inhibitor, an Akt inhibitor, an mTOR inhibitor, a binder of a T cell co-stimulatory molecule, and a chemotherapeutic agent.

20. The method according to claim 19, wherein the binder of a T cell co-stimulatory molecule is a binder of OX40.